Ifn-gamma-inducible regulatory t cell convertible anti-cancer (irtca) antibody and uses thereof

ABSTRACT

Provided are IFN-γ-Inducible Regulatory T Cell Convertible Anti-Cancer (IRTCA) antibodies and antigen-binding fragment thereof that bind to an activation-inducible TNFR (AITR) polypeptide. Various in vitro and in vivo methods and compositions related to IRTCA antibodies described herein are also provided. Methods include, for example, changing cytokine secretion from T cells in vivo or in vitro and prevention and/or therapeutic treatment of cancer using an IRTCA antibody or fragment thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application claiming priorityunder 35 U.S.C. § 111(a) to PCT Application No. PCT/I132018/000201,filed on Feb. 9, 2018, which claims priority to and the benefit of U.S.Patent Application No. 62/457,422, filed on Feb. 10, 2017. The entirecontents of the foregoing are hereby incorporated by reference.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing(submitted electronically as a .txt file named “SEQLISTING.txt” on Jan.3, 2019). The .txt file was generated on Jan. 3, 2019, and is 20,353bytes in size. The entire contents of the Sequence Listing are hereinincorporated by reference.

BACKGROUND

Cancer remains one of the leading causes of death in the world. Recentstatistics report that 13% of the world population dies from cancer.According to estimates from the International Agency for Research onCancer (IARC), in 2012 there were 14.1 million new cancer cases and 8.2million cancer deaths worldwide. By 2030, the global burden is expectedto grow to 21.7 million new cancer cases and 13 million cancer deathsdue to population growth and aging and exposure to risk factors such assmoking, unhealthy diet and physical inactivity. Further, pain andmedical expenses for cancer treatment cause reduced quality of life forboth cancer patients and their families. It is apparent that, above all,cancer is a disease for which it is necessary to urgently find improvedtreatment methods.

SUMMARY

The present disclosure provides, among other things, antibodies andfragments thereof that bind to a human activation-inducible TNFR familyreceptor (AITR) polypeptide. In some embodiments, the present inventionprovides IFN-γ-Inducible Regulatory T Cell Convertible Anti-Cancer(IRTCA) antibodies and/or antigen-binding fragments thereof, including:(a) a heavy chain CDR1 comprising a sequence of SEQ ID NO: 8 or 24, aheavy chain CDR2 comprising a sequence of SEQ ID NO: 9 or 25, and aheavy chain CDR3 comprising at least one sequence selected from SEQ IDNO: 10, 14, 15, 16, and 17, and (b) a light chain CDR1 comprising asequence of SEQ ID NO: 11, a light chain CDR2 comprising a sequence ofSEQ ID NO: 12, and a light chain CDR3 comprising a sequence of SEQ IDNO: 13 or 18, wherein the IRTCA antibody or antigen-binding fragmentthereof does not comprise each of a heavy chain CDR1 comprising asequence of SEQ ID NO: 8, a heavy chain CDR2 comprising a sequence ofSEQ ID NO: 9, a heavy chain CDR3 comprising a sequence of SEQ ID NO: 10,a light chain CDR1 comprising a sequence of SEQ ID NO: 11, a light chainCDR2 comprising a sequence of SEQ ID NO: 12 and a light chain CDR3comprising a sequence of SEQ ID NO: 13.

In some embodiments, the present invention further provides IRTCAantibodies or antigen-binding fragments wherein the antibody orantigen-binding fragment include any one of the following: (a) a heavychain variable domain comprising a sequence at least 90% identical to asequence selected from SEQ ID NOs: 3, 4, 5, 6, 20, and 21; (b) a lightchain variable domain comprising a sequence at least 90% identical to asequence selected from SEQ ID NOs: 7, 22, and 23; or (c) a heavy chainvariable domain comprising a sequence at least 90% identical to asequence selected from SEQ ID NOs: 3, 4, 5, 6, 20, and 21 and a lightchain variable domain comprising a sequence at least 98% identical to asequence selected from SEQ ID NOs: 7, 22, and 23.

In some embodiments, the present invention also provides IRTCAantibodies or antigen-binding fragments wherein the antibody orantigen-binding fragment comprises any one of the following: (a) a heavychain variable domain comprising a sequence at least 98% identical to asequence selected from SEQ ID NOs: 3, 4, 5, 6, 20, and 21; (b) a lightchain variable domain comprising a sequence at least 98% identical to asequence selected from SEQ ID NOs: 7, 22, and 23; or (c) a heavy chainvariable domain comprising a sequence at least 98% identical to asequence selected from SEQ ID NOs: 3, 4, 5, 6, 20, and 21 and a lightchain variable domain comprising a sequence at least 98% identical to asequence selected from SEQ ID NOs: 7, 22, and 23.

In some embodiments, a provided IRTCA antibody or antigen-bindingfragment includes any one of the following: (a) a heavy chain variabledomain comprising a sequence selected from SEQ ID NOs: 3, 4, 5, 6, 20,and 21; (b) a light chain variable domain comprising a sequence selectedfrom SEQ ID NOs: 7, 22, and 23; or (c) a heavy chain variable domaincomprising a sequence selected from SEQ ID NOs: 3, 4, 5, 6, 20, and 21and a light chain variable domain comprising a sequence selected fromSEQ ID NOs: 7, 22, and 23.

In some embodiments, an IRTCA antibody or antigen-binding fragmentthereof does not include each of a heavy chain CDR1 comprising asequence of SEQ ID NO: 8, a heavy chain CDR2 comprising a sequence ofSEQ ID NO: 9, a heavy chain CDR3 comprising a sequence of SEQ ID NO: 10,a light chain CDR1 comprising a sequence of SEQ ID NO: 11, a light chainCDR2 comprising a sequence of SEQ ID NO: 12 and a light chain CDR3comprising a sequence of SEQ ID NO: 13

In accordance with any of a variety of embodiments, provided IRTCAantibodies or antigen-binding fragments thereof, and/or providedcompositions may exhibit a range of binding affinities. For example, insome embodiments, a provided IRTCA antibody or antigen-binding fragmenthas a binding affinity (K_(D)) for a human Activation-Inducible TumorNecrosis Factor Receptor (TNFR) Family Receptor (AITR) molecule of1×10⁻⁷ to 1×10⁻¹² M. In some embodiments, a provided IRTCA antibody orantigen-binding fragment binds to an epitope within the extracellulardomain of human AITR polypeptide. In some embodiments, an epitope withinthe extracellular domain of human AITR polypeptide comprises SEQ ID NO:19.

In some embodiments, a provided antibody or antigen-binding fragment isor comprises a humanized antibody. In some embodiments, a provided IRTCAantibody or antigen-binding fragment thereof is or comprises amonoclonal antibody. In some embodiments, a provided IRTCA antibody orantigen-binding fragment thereof includes an immunoglobulin constantdomain, wherein the constant domain is selected from an IgG1 or avariant thereof, an IgG2 or a variant thereof, an IgG4 or a variantthereof, an IgA or a variant thereof, an IgE or a variant thereof, anIgM or a variant thereof, and an IgD or a variant thereof. In someembodiments, a provided IRTCA antibody or antigen-binding fragmentthereof is or comprises a human IgG1. In some embodiments, the IgG1 isor comprises a sequence that is at least 95% identical to SEQ ID NO: 26.In some embodiments, a provided IRTCA antibody or antigen-bindingfragment thereof is or comprises a Fab fragment, a Fab′ fragment, aF(ab′)2 fragment, a Fv fragment, a disulfide-bonded Fv fragment, a scFvfragment, a single domain antibody, humabody, nanobody, or a diabody.

In accordance with various embodiments, the present invention provides avariety of molecules and compositions for, inter alia, facilitatingdelivery and/or expression of a provided composition that comprises anamount of a provided IRTCA antibody or antigen-binding fragment thereof.For example, in some embodiments, the present invention provides nucleicacid molecules encoding an IRTCA antibody or antigen-binding fragmentthereof. In some embodiments, the present invention also providesrecombinant vectors including such nucleic acid molecules. In someembodiments, the present invention also provides host cells including aprovided recombinant vector and/or a provided nucleic acid molecule. Insome embodiments, a host cell may be selected from a bacterial, yeast,insect or mammalian cell. In some embodiments a host cell is selectedfrom the group consisting of E. coli, P. pastoris, Sf9, COS, HEK293,Expi293, CHO-S, CHO-DG44, CHO-K1, and a mammalian lymphocyte.

In some embodiments, the present invention provides pharmaceuticalcompositions including: (a) one or more provided IRTCA antibodies orantigen-binding fragments thereof, one or more provided nucleic acidmolecules, one or more provided recombinant vectors, and/or one or moreprovided host cells, and (b) a pharmaceutically acceptable carrier. Anyof a variety of pharmaceutically acceptable carriers may be used inaccordance with various embodiments.

In addition to the variety of powerful new antibodies, antigen-bindingfragments thereof, and compositions provided herein, the presentinvention also provides, in various embodiments, a variety of newtherapeutic methods as well. For example, and in accordance with variousembodiments, the present invention provides methods of treating asubject in need thereof, the method including the step of: administeringto the subject a composition that comprises or delivers a provided IRTCAantibody or antigen-binding fragment thereof, a provided nucleic acidmolecule, and/or a provided recombinant vector.

By way of additional example, the present invention also provides, insome embodiments, methods of inducing an immune response in a subject inneed thereof, the method including the step of: administering to thesubject a composition that comprises or delivers a provided IRTCAantibody or antigen-binding fragment thereof, a provided nucleic acidmolecule, and/or a provided recombinant vector.

By way of further example, in some embodiments, the present inventionalso provides methods of enhancing an immune response or increasing theactivity of an immune cell in a subject in need thereof, the methodincluding the step of: administering to the subject a composition thatcomprises or delivers a provided IRTCA antibody or antigen-bindingfragment thereof, a provided nucleic acid molecule, and/or a providedrecombinant vector.

In some embodiments, the present invention also provides methods forincreasing secretion of IFN-γ by a T cell in vivo or in vitro, themethod comprising: contacting the cell with a composition that comprisesor delivers an amount of a provided IRTCA antibody or antigen-bindingfragment thereof. In some embodiments, the present invention alsoprovides methods for decreasing secretion of TGF-β by a T cell in vivoor in vitro, the method comprising: contacting the cell with acomposition that comprises or delivers an amount of a provided IRTCAantibody or antigen-binding fragment thereof. In some embodiments, thepresent invention also provides methods for converting a T cell into aType 1 helper T (T_(H)1) cell, the method comprising: contacting thecell with a composition that comprises or delivers an amount of aprovided IRTCA antibody or antigen-binding fragment thereof.

In some embodiments, the subject has, or is at risk for developing,cancer. In some embodiments, the cancer is selected from a bladdercancer, breast cancer, cervical cancer, colon cancer, endometrialcancer, esophageal cancer, fallopian tube cancer, gall bladder cancer,gastrointestinal cancer, head and neck cancer, hematological cancer,laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma,mesothelioma, ovarian cancer, primary peritoneal cancer, salivary glandcancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer,renal cell carcinoma, glioblastoma, and prostate cancer.

It is specifically contemplated that various embodiments are suitablefor us in one or more combination therapy regimen. For example, in someembodiments, the subject has been administered or will be administeredone or more additional anticancer therapies. In some embodiments, theone or more additional cancer therapies is selected from ionizingradiation, a chemotherapeutic agent, an antibody agent, and a cell-basedtherapy, such that the subject receives treatment with both. In someembodiments, the one or more additional anticancer therapies comprise animmune checkpoint inhibitor, IL-12, GM-CSF, an anti-CD4 agent,cisplatin, fluorouracil, doxorubicin, irinotecan, paclitaxel,indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor, or cyclophosphamide.

Various embodiments of the present invention may also be useful fordetermining an advantageous dosing regimen for one or more providedIRTCA antibodies and/or antigen binding fragments thereof, nucleic acidmolecules, recombinant vectors, and/or host cells. For example, in someembodiments, the present invention provides methods of determining adose of an IRTCA antibody or antigen binding fragment thereof fortherapeutic treatment of a subject in need thereof, the method includingthe steps of: (a) providing or obtaining a measurement of secreted IFN-γin a biological sample from the subject, wherein the subject has beenadministered a composition that comprises or delivers an amount of aprovided IRTCA antibody or antigen-binding fragment thereof, and (b)comparing the measurement of secreted IFN-γ to a reference value,wherein if the measurement of secreted IFN-γ is higher or lower than thereference value, adjusting the amount of the IRTCA antibody or antigenbinding fragment thereof to be administered, thereby determining a dosefor therapeutic treatment of a subject.

In some embodiments, a reference value may be a level of IFN-γ in abiological sample from the subject prior to administration of theprovided IRTCA antibody or antigen-binding fragment thereof. In someembodiments, if a measured amount of secreted IFN-γ in a biologicalsample from the subject is higher than a reference value, then treatmentis either maintained at the same level given previously, or treatment isceased for a period of time.

By way of additional example, in some embodiments, the present inventionprovides methods of determining a dose of an IRTCA antibody or antigenbinding fragment thereof for therapeutic treatment of a subject in needthereof, including the steps of: (a) providing or obtaining ameasurement of T_(reg) cell population in a biological sample from thesubject, wherein the subject has been administered a composition thatcomprises or delivers an amount of a provided IRTCA antibody orantigen-binding fragment thereof; and (b) comparing the measurement ofT_(reg) cell population to a reference value, wherein if the measurementof T_(reg) cell population is higher or lower than the reference value,adjusting the amount of the IRTCA antibody or antigen binding fragmentthereof to be administered, thereby determining a dose for therapeutictreatment of a subject. In some embodiments, a reference value may be alevel of T_(reg) cell population in a biological sample from the subjectprior to administration of the provided IRTCA antibody orantigen-binding fragment thereof. In some embodiments, if a measuredamount of T_(reg) cell population in a biological sample from thesubject is lower than a reference value, then treatment is eithermaintained at the same level given previously, or treatment is ceasedfor a period of time.

By way of additional example, in some embodiments, the present inventionprovides methods of determining a dose of an IRTCA antibody or antigenbinding fragment thereof for therapeutic treatment of a subject in needthereof, including the steps of: (a) providing or obtaining ameasurement of IFN-γ-secreting T cell population in a biological samplefrom the subject, wherein the subject has been administered acomposition that comprises or delivers an amount of a provided IRTCAantibody or antigen-binding fragment thereof; (b) providing or obtaininga measurement of T_(reg) cell population in a biological sample from thesubject, wherein the subject has been administered a composition thatcomprises or delivers an amount of a provided IRTCA antibody orantigen-binding fragment thereof; (c) calculating the ratio of themeasurement of IFN-γ-secreting T cell population to the measurement ofT_(reg) cell population; and (d) comparing the ratio to a referencevalue, wherein if ratio is higher or lower than the reference value,adjusting the amount of the IRTCA antibody or antigen binding fragmentthereof to be administered, thereby determining a dose for therapeutictreatment of a subject. In some embodiments, a reference value may be aratio of a level of IFN-γ-secreting T cell population in a biologicalsample from a subject to a level of T_(reg) cell population in the samebiological sample from the subject prior to administration of theprovided IRTCA antibody or antigen-binding fragment thereof. In someembodiments, if a calculated ratio in a biological sample from thesubject is higher than a reference value, then treatment is eithermaintained at the same level given previously, or treatment is ceasedfor a period of time.

In some embodiments, a reference value comprises an index value whichincludes a value derived from one or more healthy subjects or a valuederived from one or more cancer-diagnosed subjects. In some embodiments,a biological sample is a sample of whole blood, plasma, tumor tissue, orserum.

In some embodiments, the present invention also provides methods ofvalidating affinity of an antibody or antigen-binding fragment thereoffor AITR, the method including the steps of: contacting a T cell with acomposition that comprises or delivers an amount of a provided IRTCAantibody or antigen-binding fragment thereof, and measuring cytokinesecretion from the T cell, wherein cytokine secretion correlates withimmune response enhancement, anti-cancer effects and/or anti-tumoreffects of the IRTCA antibody. In some embodiments, the T cell expressesAITR protein. In some embodiments, the T cell is a regulatory T cell(T_(reg) cell). In some embodiments, the T cell is an effector T cell(T_(eff) cell).

In accordance with any of a variety of embodiments, provided IRTCAantibodies or antigen-binding fragments thereof may bind to at least oneof the following residues within the extracellular domain of the humanAITR peptide: H1, C2, G3, D4, P5, C6, C7, T8, T9, and C10 of SEQ ID NO:19, when the antibody or the antigen-binding fragment bound to a humanAITR peptide.

In some embodiments, a provided antibody or antigen-binding fragment maybind to at least two, at least three, at least four, or at least five ofthe following residues within the extracellular domain of the human AITRpeptide: H1, C2, G3, D4, P5, C6, C7, T8, T9, and C10 of SEQ ID NO: 19,when the antibody or the antigen-binding fragment is bound to a humanAITR peptide.

In some embodiments, a provided antibody or antigen-binding fragment maybinds to at least one, at least two, at least three, at least four, orall five of the following residues within the extracellular domain ofthe human AITR peptide: C6, C7, T8, T9, C10 of SEQ ID NO: 19, when theantibody or the antigen-binding fragment is bound to a human AITRpeptide.

In some embodiments, a provided antibody or antigen-binding fragment maybinds to at least one of the following residues within the extracellulardomain of the human AITR peptide: E1, C2, C3, S4, E5, W6, D7, C8, M9,and C10 of SEQ ID NO: 28, when the antibody or the antigen-bindingfragment is bound to a human AITR peptide.

In some embodiments, a provided antibody or antigen-binding fragment maybinds to at least two, at least three, at least four, or at least fiveof the following residues within the extracellular domain of the humanAITR peptide: E1, C2, C3, S4, E5, W6, D7, C8, M9, and C10 of SEQ ID NO:28, when the antibody or the antigen-binding fragment is bound to ahuman AITR peptide.

In some embodiments, a provided antibody or antigen-binding fragment maybinds to at least one, at least two, at least three, at least four, orall five of the following residues within the extracellular domain ofthe human AITR peptide: C2, C3, S4, E5, and W6 of SEQ ID NO: 28, whenthe antibody or the antigen-binding fragment is bound to a human AITRpeptide.

In some embodiments, a provided antibody or antigen-binding fragment maybinds to at least one of the following residues within the extracellulardomain of the human AITR peptide: G1, T2, D3, A4, R5, C6, C7, R8, V9,H10, and T11 of SEQ ID NO: 30, when the antibody or the antigen-bindingfragment is bound to a human AITR peptide.

In some embodiments, a provided antibody or antigen-binding fragment maybinds to at least two, at least three, at least four, or at least fiveof the following residues within the extracellular domain of the humanAITR peptide: G1, T2, D3, A4, R5, C6, C7, R8, V9, H10, and T11 of SEQ IDNO: 30, when the antibody or the antigen-binding fragment is bound to ahuman AITR peptide.

In some embodiments, a provided antibody or antigen-binding fragment maybinds to at least one, at least two, at least three, at least four, orall five of the following residues within the extracellular domain ofthe human AITR peptide: C6, C7, R8, V9, and H10 of SEQ ID NO: 30.

In some embodiments, a provided antibody or antigen-binding fragment mayinclude one or more amino acid mutation(s) or modification(s) ascompared to a parent antibody or antigen-binding fragment, wherein themutation(s) or modification(s) result in the antibody or antibodyfragment having reduced tendency to aggregate.

In some embodiments, a provided antibody or antigen-binding fragment mayinclude mutation(s) or modification(s) that result in changes to theisoelectric point (pI) of the antibody or antigen-binding fragment ascompared to a parent antibody or antibodyantigen-binding fragment.

In some embodiments a provided antibody or antigen-binding fragment mayinclude mutation(s) or modification(s) that result in a change of about−5, −4, −3, −2, −1, −0.7, −0.5, −0.4, −0.3, −0.2, −0.1, +0.1, +0.2,+0.3, +0.4, +0.5, +0.7, +1, +2, +3, +4, or +5 in pI of the antibody orantibody fragment as compared to a parent antibody orantibodyantigen-binding fragment.

In some embodiments the change in pI of a provided antibody orantigen-binding fragment may be measured using isoelectric focusing.

In some embodiments a provided antibody or antigen-binding fragment mayinclude mutation(s) or modification(s) that result in an antibody orantigen-binding fragment having an improved thermodynamic stability.

In some embodiments the improved thermodynamic stability of a providedantibody or antigen-binding fragment may be measured by an increase inaggregation onset temperature (T_(agg)) or thermal unfolding temperature(T_(m)).

In some embodiments a provided antibody or antigen-binding fragment mayinclude mutation(s) or modification(s) that result in at least a 0.1°C., 0.2° C., 0.3° C., 0.4° C., 0.5° C., 0.7° C., 1° C., 1.5° C., 2° C.,3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C.,13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C., or 20° C.increase in T_(m) or T_(agg) of the antibody or antigen-binding fragmentas compared to the T_(m) or T_(agg) of a parent antibody orantigen-binding fragment.

In some embodiments the T_(m) or T_(agg) of a provided antibody orantigen-binding fragment may be measured by differential scanningcalorimetry.

In some embodiments a provided antibody or antigen-binding fragment mayinclude mutation(s) or modification(s) that result in at least a 5%,10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% reduction in aggregateformation as compared to a parent antibody or antigen-binding fragment.

In some embodiments the reduction in aggregate formation of a providedantibody or antigen-binding fragment may be measured by size exclusionchromatography or dynamic light scattering assays.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any citations to publications, patents, or patentapplications herein are incorporated by reference in their entirety. Anynumerals used in this application with or without about/approximatelyare meant to cover any normal fluctuations appreciated by one ofordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present invention, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE DRAWING

The Drawing included herein, which is comprised of the followingFigures, is for illustration purposes only and not for limitation. Theforegoing and other objects, aspects, features, and advantages of thepresent disclosure will become more apparent and better understood byreferring to the following description taken in conjunction with theaccompanying figures in which:

FIGS. 1A-1B show Fab type amino acid sequences of IRTCA-A, wherein FIG.1A and FIG. 1B respectively represent the amino acid sequence of theIRTCA-A Fab type and the IRTCA-A amino acid sequence into which a stopcodon is inserted (asterisk indicates stop codon).

FIGS. 2A-2B shows the amino acid regions of IRTCA-A in which randomizedPCR configuration and mutation were induced. FIG. 2A shows the IRTCA-Alight chain, and FIG. 2B shows the IRTCA-A heavy chain. A random primerwas used so that mutation might be induced at the CDR3 region, and aphage display library where mutation was concentrated in the CDR3 areawas fabricated.

FIG. 3 shows the dissociation curve overlay plot for the AITR antigen of16 heavy chain mutants and 9 light chain mutants in the IRTCA-A series.

FIG. 4 shows the dissociation curve overlay plot for the AITR antigen of22 light chain mutants in the IRTCA-A series.

FIG. 5 shows a graph of secretion of cytokine IFN-γ by IRTCA-A in CD4⁺ Tcells. IRTCA-A induces IFN-γ secretion in a dose dependent manner.

FIG. 6A depicts a graph quantifying dose-dependent changes in secretionof IFN-γ from T_(reg) cells after treatment with IRTCA-A. FIG. 6Bdepicts a graph quantifying dose-dependent changes in secretion of TGF-βfrom T_(reg) cells after treatment with IRTCA-A.

FIG. 7A depicts a graph quantifying dose-dependent changes in secretionof IFN-γ in CD4 T cells after treatment with several IRTCA-A mutants.FIG. 7B depicts a graph quantifying dose-dependent changes in secretionof IFN-γ in T_(reg) cells after treatment with several IRTCA-A mutants.FIG. 7C depicts a graph quantifying dose-dependent changes in secretionof TGF-β in T_(reg) cells after treatment with several IRTCA-A mutants.

FIG. 8 depicts graphs showing dose-dependent reductions in tumor volume(from injection with colorectal cancer cells) following treatment ofHu-PBL-SCID mice with IRTCA antibody.

FIG. 9 depicts a graph quantifying the effect of control hIgG, H1F1antibody, MK4166 and Keytruda on tumor size. Arrows indicate days ofinjection with treatment or control antibody.

FIG. 10A depicts a graph quantifying the effect of H1F1 or controlantibody on tumor size (triple negative breast cancer) after 3 doses(arrows) of antibody treatment. FIG. 10B depicts a graph quantifying theeffect of H1F1 or control antibody on tumor size (colon cancer) after 3doses (arrows) of antibody treatment. FIG. 10C depicts a graphquantifying the effect of H1F1 or control antibody on tumor sizemelanoma) after 3 doses (arrows) of antibody treatment. FIG. 10D depictsa graph quantifying the effect of H1F1 or control antibody on tumor size(melanoma) after 3 doses (arrows) of antibody treatment.

FIG. 11 depicts graphs quantifying the effect of H1F1 on cytokinesecretion in CD4⁺CD25^(high)Foxp3⁺ T cells of Macaca fascicularis.

FIGS. 12A-12D depict graphs quantifying H1F1-mediated conversion ofregulatory T cells to T_(H)1 cells in macaques in vivo. Unlike thehIgG-treated control group (FIG. 12A), the cells treated with 22 mg/kgH1F1 (FIG. 12B), 45 mg/kg H1F1 (FIG. 12C) and 90 mg/kg H1F1 (FIG. 12D)showed an increase in IFN-γ positive CD4⁺T cells over the course of 3weeks.

FIGS. 13A-13D depict graphs showing that cytokine induction by H1F1 wasdifferential. Secretion of the cytokines IL-4 (FIG. 13B), and IL-5 (FIG.13C) was not effected in cells treated with H1F1, whereas IL-2 (FIG.13A) and IFN-γ secretion (FIG. 13D) increased after treatment with H1F1.

FIGS. 14A-14D depict graphs quantifying the dose-dependent effect ofH1F1 on populations of cells expressing CD4⁺CD25⁺Foxp3^(high) (FIG.14A), TGF-β (FIG. 14B), CD4⁺IFN-γ (FIG. 14C) and CD4⁺T-bet⁺ (FIG. 14D).

FIG. 15 depicts H1F1-specific staining in EBV-positive gastric cancercells and bone marrow from an AML patient.

FIG. 16A depicts binding of H1F1 to AITR-5 at concentrations of 10, 5,2.5 and 1.25 μg/mL, as compared to the negative control mAb EU101, whichdid not bind to AITR-5. FIG. 16B depicts binding of H1F1M69 and H1F1M74to AITR-5 in a dose dependent manner.

FIGS. 17A-C depict the conversion of nT_(reg) cells to T_(H)1-like cellsupon stimulation with H1F1M69 (FIG. 17B) and H1F1M74 (FIG. 17C) relativeto an hIgG control (FIG. 17A). The numbers in each panel indicate thepercentage of positive cells.

FIG. 18 depicts a graph quantifying the dose-dependent effect of H1F1M69and H1F1M74 on TGF-β and IFN-γ in nT_(reg) cells.

FIG. 19 depicts the generation of iT_(reg) cells from CD4⁺ T cells afterstimulation with anti-CD3, CD28 beads, IL-2 and TGF-β for 6 days.

FIGS. 20A-20G depict the binding efficiency of H1F1M69 and H1F1M74 forAITR on iT_(reg) cells. FIGS. 20A and 20B illustrate gating parameters.FIG. 20C illustrates an isotype control. FIGS. 20D-20G show that surfaceAITR was detected by H1F1M69 (FIG. 20D) and H1F1M74 (FIG. 20E) morefrequently than by competitor's anti-AITR antibodies TRX518 (FIG. 20F)and MK4166 (FIG. 20G).

FIGS. 21A-21E depicts the conversion of iT_(reg) cells to T_(H)1-likecells upon stimulation anti-AITR antibodies. FIG. 21A shows an hIgGcontrol. FIGS. 21B-21E show the effect of H1F1M69 (FIG. 21B), H1F1M74(FIG. 21C), TRX518 (FIG. 21D) and MK4166 (FIG. 21E) on iT_(reg) cells.

FIG. 22 depicts a graph quantifying the dose-dependent effect of H1F1M69and H1F1M74 on TGF-β and IFN-γ in iT_(reg) cells.

FIGS. 23A-23E depict the effect of immobilized monoclonal antibodieshIgG (FIG. 23A), H1F1M69 (FIG. 23B), H1F1M74 (FIG. 23C), TRX518 (FIG.23D) and MK4166 (FIG. 23E) on Foxp3 in iT_(reg) cells.

FIGS. 24A-24B depict the conversion of iT_(reg) cells to T_(H)1-likecells after being added to wells coated with IL-2 and H1F1M69 (FIG. 24A)or H1F1M74 (FIG. 24B). The numbers in each panel indicate the percentageof positive cells.

FIGS. 25A-25C depict the polarization of T_(eff) cells to T_(H)1 cellsafter treatment with antibodies. FIG. 25A shows the effect of a controlhIgG. FIG. 25B shows the effect of H1F1M69 and H1F1M74. FIG. 25C showsthe effect of antibodies TRX518 and MK4166.

FIG. 26 depicts a graph quantifying the dose-dependent effect of H1F1M69and H1F1M74 on IFN-γ in T_(eff) cells.

FIG. 27A depicts a full-length AITR polypeptide and twelve GST fusionprotein constructs containing AITR deletion mutants, R1-R12.

FIG. 27B depicts binding of the anti-AITR antibodies IRTCA-A (A27),H1F1, H1F1M69, and H1F1M74 to the GST fusion protein constructs R1-R12(shown in FIG. 27A) containing AITR deletion mutants, as shown byWestern blot analysis (left panels) and ELISA (right panels).

FIG. 27C depicts the binding epitope (underlined) for IRTCA-A (A27),H1F1, H1F1M69, and H1F1M74, that was identified by the Western blot andELISA analysis of FIG. 27B.

FIG. 28A depicts a full-length AITR polypeptide and fourteen mutantpeptide constructs spanning the binding epitope identified in FIG. 27C.Each mutant peptide construct contains a C-terminal Histidine tag and analanine substitution at one amino acid of the binding epitope, R1-R14.

FIG. 28B depicts binding of the anti-AITR antibodies IRTCA-A (A27),H1F1, H1F1M69, and H1F1M74 to the mutant peptide constructs shown inFIG. 28A, as shown by Western blot analysis (left panels) and ELISA(right panels).

FIG. 28C depicts the binding epitopes (underlined) and core residues(indicated by asterisks above the residues) for IRTCA-A (A27), H1F1,H1F1M69 and H1F1M74, that were identified by Western blot and ELISAanalysis of FIGS. 27B and 28B.

FIG. 29A depicts a full-length AITR polypeptide and twelve GST fusionprotein constructs containing AITR deletion mutants, R1-R12.

FIG. 29B depicts binding of the anti-AITR antibodies IRTCA-A (A27), A35,and A41 to the GST fusion protein constructs R1 to R12 (shown in FIG.29A), as shown by Western blot analysis.

FIG. 29C depicts the binding epitopes (underlined) for IRTCA-A (A27),A35, and A41, that were identified by the Western blot analysis of FIG.29B.

FIG. 30A depicts three His-fusion constructs to which analine mutationswere individually introduced at each of the epitope residues for IRTCA-A(A27), A35, and A41 to create His fusion constructs containing alaninesubstituted AITR extracellular domains within the extracellular domain.

FIG. 30B depicts binding of the anti-AITR antibodies IRTCA-A (A27), A35,and A41 to His fusion constructs containing alanine substituted AITRextracellular domains, as shown by Western blot analysis.

FIG. 30C depicts binding of the anti-AITR antibodies IRTCA-A (A27), A35,and A41 to His fusion constructs containing alanine substituted AITRextracellular domains as shown by ELISA analysis.

CERTAIN DEFINITIONS

In the description that follows, a number of terms used in recombinantDNA and immunology are extensively utilized. In order to provide aclearer and consistent understanding of the specification and claims,including the scope to be given such terms, the following definitionsare provided.

About: The term “about”, when used herein in reference to a value,refers to a value that is similar, in context to the referenced value.In general, those skilled in the art, familiar with the context, willappreciate the relevant degree of variance encompassed by “about” inthat context. For example, in some embodiments, the term “about” mayencompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%,15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, orless of the referred value.

Administration: As used herein, the term “administration” typicallyrefers to the administration of a composition to a subject or system toachieve delivery of an agent that is, or is included in, thecomposition. Those of ordinary skill in the art will be aware of avariety of routes that may, in appropriate circumstances, be utilizedfor administration to a subject, for example a human. For example, insome embodiments, administration may be ocular, oral, parenteral,topical, etc. In some particular embodiments, administration may bebronchial (e.g., by bronchial instillation), buccal, dermal (which maybe or comprise, for example, one or more of topical to the dermis,intradermal, interdermal, transdermal, etc.), enteral, intra-arterial,intradermal, intragastric, intramedullary, intramuscular, intranasal,intraperitoneal, intrathecal, intravenous, intraventricular, within aspecific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal,subcutaneous, sublingual, topical, tracheal (e.g., by intratrachealinstillation), vaginal, vitreal, etc. In some embodiments,administration may involve only a single dose. In some embodiments,administration may involve application of a fixed number of doses. Insome embodiments, administration may involve dosing that is intermittent(e.g., a plurality of doses separated in time) and/or periodic (e.g.,individual doses separated by a common period of time) dosing. In someembodiments, administration may involve continuous dosing (e.g.,perfusion) for at least a selected period of time.

Affinity: As is known in the art, “affinity” is a measure of thetightness with a particular ligand binds to its partner. Affinities canbe measured in different ways. In some embodiments, affinity is measuredby a quantitative assay. In some such embodiments, binding partnerconcentration may be fixed to be in excess of ligand concentration so asto mimic physiological conditions. Alternatively or additionally, insome embodiments, binding partner concentration and/or ligandconcentration may be varied. In some such embodiments, affinity may becompared to a reference under comparable conditions (e.g.,concentrations).

Agonist: Those skilled in the art will appreciate that the term“agonist” may be used to refer to an agent condition, or event whosepresence, level, degree, type, or form correlates with an increasedlevel of activity of another agent (i.e., the agonized agent). Ingeneral, an agonist may be or include an agent of any chemical classincluding, for example, small molecules, polypeptides, nucleic acids,carbohydrates, lipids, metals, and/or any other entity that shows therelevant activating activity. In some embodiments, an agonist may bedirect (in which case it exerts its influence directly upon its target);in some embodiments, an agonist may be indirect (in which case it exertsits influence by other than binding to its target; e.g., by interactingwith a regulator of the target, so that level or activity of the targetis altered).

Animal: as used herein refers to any member of the animal kingdom. Insome embodiments, “animal” refers to humans, of either sex and at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal, geneticallyengineered animal, and/or a clone.

Antagonist: Those skilled in the art will appreciate that the term“antagonist”, as used herein, may be used to refer to an agentcondition, or event whose presence, level, degree, type, or formcorrelates with decreased level or activity of another agent (i.e., theinhibited agent, or target). In general, an antagonist may be or includean agent of any chemical class including, for example, small molecules,polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or anyother entity that shows the relevant inhibitory activity. In someembodiments, an antagonist may be direct (in which case it exerts itsinfluence directly upon its target); in some embodiments, an antagonistmay be indirect (in which case it exerts its influence by other thanbinding to its target; e.g., by interacting with a regulator of thetarget, so that level or activity of the target is altered).

Antibody: As used herein, the term “antibody” refers to a polypeptidethat includes canonical immunoglobulin sequence elements sufficient toconfer specific binding to a particular target antigen. As is known inthe art, intact antibodies as produced in nature are approximately 150kD tetrameric agents comprised of two identical heavy chain polypeptides(about 50 kD each) and two identical light chain polypeptides (about 25kD each) that associate with each other into what is commonly referredto as a “Y-shaped” structure. Each heavy chain is comprised of at leastfour domains (each about 110 amino acids long)—an amino-terminalvariable (VH) domain (located at the tips of the Y structure), followedby three constant domains: CH1, CH2, and the carboxy-terminal CH3(located at the base of the Y's stem). A short region, known as the“switch”, connects the heavy chain variable and constant regions. The“hinge” connects CH2 and CH3 domains to the rest of the antibody. Twodisulfide bonds in this hinge region connect the two heavy chainpolypeptides to one another in an intact antibody. Each light chain iscomprised of two domains—an amino-terminal variable (VL) domain,followed by a carboxy-terminal constant (CL) domain, separated from oneanother by another “switch”. Intact antibody tetramers are comprised oftwo heavy chain-light chain dimers in which the heavy and light chainsare linked to one another by a single disulfide bond; two otherdisulfide bonds connect the heavy chain hinge regions to one another, sothat the dimers are connected to one another and the tetramer is formed.Naturally-produced antibodies are also glycosylated, typically on theCH2 domain. Each domain in a natural antibody has a structurecharacterized by an “immunoglobulin fold” formed from two beta sheets(e.g., 3-, 4-, or 5-stranded sheets) packed against each other in acompressed antiparallel beta barrel. Each variable domain contains threehypervariable loops known as “complement determining regions” (CDR1,CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1,FR2, FR3, and FR4). When natural antibodies fold, the FR regions formthe beta sheets that provide the structural framework for the domains,and the CDR loop regions from both the heavy and light chains arebrought together in three-dimensional space so that they create a singlehypervariable antigen binding site located at the tip of the Ystructure. The Fc region of naturally-occurring antibodies binds toelements of the complement system, and also to receptors on effectorcells, including for example effector cells that mediate cytotoxicity.As is known in the art, affinity and/or other binding attributes of Fcregions for Fc receptors can be modulated through glycosylation or othermodification. In some embodiments, antibodies produced and/or utilizedin accordance with the present invention include glycosylated Fcdomains, including Fc domains with modified or engineered suchglycosylation. For purposes of the present invention, in certainembodiments, any polypeptide or complex of polypeptides that includessufficient immunoglobulin domain sequences as found in naturalantibodies can be referred to and/or used as an “antibody”, whether suchpolypeptide is naturally produced (e.g., generated by an organismreacting to an antigen), or produced by recombinant engineering,chemical synthesis, or other artificial system or methodology. In someembodiments, an antibody is polyclonal; in some embodiments, an antibodyis monoclonal. In some embodiments, an antibody has constant regionsequences that are characteristic of mouse, rabbit, primate, or humanantibodies. In some embodiments, antibody sequence elements arehumanized, primatized, chimeric, etc., as is known in the art. Moreover,the term “antibody” as used herein, can refer in appropriate embodiments(unless otherwise stated or clear from context) to any of the art-knownor developed constructs or formats for utilizing antibody structural andfunctional features in alternative presentation. For example,embodiments, an antibody utilized in accordance with the presentinvention is in a format selected from, but not limited to, intact IgA,IgG, IgE or IgM antibodies; bi- or multi-specific antibodies (e.g.,Zybodies®, etc.); antibody fragments such as Fab fragments, Fab′fragments, F(ab′)2 fragments, Fd′ fragments, Fd fragments, and isolatedCDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; singledomain antibodies (e.g., shark single domain antibodies such as IgNAR orfragments thereof); cameloid antibodies; masked antibodies (e.g.,Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™”); singlechain or Tandem diabodies (TandAb®); humabodies, VHHs; Anticalins®;Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®;Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®;Trans-Bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®;Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack acovalent modification (e.g., attachment of a glycan) that it would haveif produced naturally. In some embodiments, an antibody may contain acovalent modification (e.g., attachment of a glycan, a payload [e.g., adetectable moiety, a therapeutic moiety, a catalytic moiety, etc.], orother pendant group [e.g., poly-ethylene glycol, etc.]

Antibody fragment: As used herein, an “antibody fragment” refers to aportion of an antibody or antibody agent as described herein, andtypically refers to a portion that includes an antigen-binding portionor variable region thereof. An antibody fragment may be produced by anymeans. For example, in some embodiments, an antibody fragment may beenzymatically or chemically produced by fragmentation of an intactantibody or antibody agent. Alternatively, in some embodiments, anantibody fragment may be recombinantly produced (i.e., by expression ofan engineered nucleic acid sequence. In some embodiments, an antibodyfragment may be wholly or partially synthetically produced. In someembodiments, an antibody fragment (particularly an antigen-bindingantibody fragment) may have a length of at least about 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 amino acids ormore, in some embodiments at least about 200 amino acids.

Binding: It will be understood that the term “binding”, as used herein,typically refers to a non-covalent association between or among two ormore entities. “Direct” binding involves physical contact betweenentities or moieties; indirect binding involves physical interaction byway of physical contact with one or more intermediate entities. Bindingbetween two or more entities can typically be assessed in any of avariety of contexts—including where interacting entities or moieties arestudied in isolation or in the context of more complex systems (e.g.,while covalently or otherwise associated with a carrier entity and/or ina biological system or cell).

Cancer: The terms “cancer”, “malignancy”, “neoplasm”, “tumor”, and“carcinoma”, are used herein to refer to cells that exhibit relativelyabnormal, uncontrolled, and/or autonomous growth, so that they exhibitan aberrant growth phenotype characterized by a significant loss ofcontrol of cell proliferation. In some embodiments, a tumor may be orcomprise cells that are precancerous (e.g., benign), malignant,pre-metastatic, metastatic, and/or non-metastatic. The presentdisclosure specifically identifies certain cancers to which itsteachings may be particularly relevant. In some embodiments, a relevantcancer may be characterized by a solid tumor. In some embodiments, arelevant cancer may be characterized by a hematologic tumor. In general,examples of different types of cancers known in the art include, forexample, hematopoietic cancers including leukemias, lymphomas (Hodgkin'sand non-Hodgkin's), myelomas and myeloproliferative disorders; sarcomas,melanomas, adenomas, carcinomas of solid tissue, squamous cellcarcinomas of the mouth, throat, larynx, and lung, liver cancer,genitourinary cancers such as prostate, cervical, bladder, uterine, andendometrial cancer and renal cell carcinomas, bone cancer, pancreaticcancer, skin cancer, cutaneous or intraocular melanoma, cancer of theendocrine system, cancer of the thyroid gland, cancer of the parathyroidgland, head and neck cancers, breast cancer, gastro-intestinal cancersand nervous system cancers, benign lesions such as papillomas, and thelike.

CDR: as used herein, refers to a complementarity determining regionwithin an antibody variable region. There are three CDRs in each of thevariable regions of the heavy chain and the light chain, which aredesignated CDR1, CDR2 and CDR3, for each of the variable regions. A “setof CDRs” or “CDR set” refers to a group of three or six CDRs that occurin either a single variable region capable of binding the antigen or theCDRs of cognate heavy and light chain variable regions capable ofbinding the antigen. Certain systems have been established in the artfor defining CDR boundaries (e.g., Kabat, Chothia, etc.); those skilledin the art appreciate the differences between and among these systemsand are capable of understanding CDR boundaries to the extent requiredto understand and to practice the claimed invention.

Chemotherapeutic Agent: The term “chemotherapeutic agent”, has usedherein has its art-understood meaning referring to one or morepro-apoptotic, cytostatic and/or cytotoxic agents, for examplespecifically including agents utilized and/or recommended for use intreating one or more diseases, disorders or conditions associated withundesirable cell proliferation. In many embodiments, chemotherapeuticagents are useful in the treatment of cancer. In some embodiments, achemotherapeutic agent may be or comprise one or more alkylating agents,one or more anthracyclines, one or more cytoskeletal disruptors (e.g.microtubule targeting agents such as taxanes, maytansine and analogsthereof, of), one or more epothilones, one or more histone deacetylaseinhibitors HDACs), one or more topoisomerase inhibitors (e.g.,inhibitors of topoisomerase I and/or topoisomerase II), one or morekinase inhibitors, one or more nucleotide analogs or nucleotideprecursor analogs, one or more peptide antibiotics, one or moreplatinum-based agents, one or more retinoids, one or more vincaalkaloids, and/or one or more analogs of one or more of the following(i.e., that share a relevant anti-proliferative activity). In someparticular embodiments, a chemotherapeutic agent may be or comprise oneor more of Actinomycin, All-trans retinoic acid, an Auiristatin,Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin,Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Curcumin,Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin,Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine,Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine and/or analogsthereof (e.g. DM1) Mechlorethamine, Mercaptopurine, Methotrexate,Mitoxantrone, a Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed,Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine,Vindesine, Vinorelbine, and combinations thereof. In some embodiments, achemotherapeutic agent may be utilized in the context of anantibody-drug conjugate. In some embodiments, a chemotherapeutic agentis one found in an antibody-drug conjugate selected from the groupconsisting of: hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38,hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox,hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox,hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4/D10-doxorubicin, gemtuzumabozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumabozogamicin, glembatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062,SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME,ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593,RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-529,vorsetuzumab mafodotin, and lorvotuzumab mertansine.

Corresponding to: As used herein, the term “corresponding to” may beused to designate the position/identity of a structural element in acompound or composition through comparison with an appropriate referencecompound or composition. For example, in some embodiments, a monomericresidue in a polymer (e.g., an amino acid residue in a polypeptide or anucleic acid residue in a polynucleotide) may be identified as“corresponding to” a residue in an appropriate reference polymer. Forexample, those of ordinary skill will appreciate that, for purposes ofsimplicity, residues in a polypeptide are often designated using acanonical numbering system based on a reference related polypeptide, sothat an amino acid “corresponding to” a residue at position 190, forexample, need not actually be the 190^(th) amino acid in a particularamino acid chain but rather corresponds to the residue found at 190 inthe reference polypeptide; those of ordinary skill in the art readilyappreciate how to identify “corresponding” amino acids. For example,those skilled in the art will be aware of various sequence alignmentstrategies, including software programs such as, for example, BLAST,CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER,HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST,PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS,SWIMM, or SWIPE that can be utilized, for example, to identify“corresponding” residues in polypeptides and/or nucleic acids inaccordance with the present disclosure.

Engineered: In general, the term “engineered” refers to the aspect ofhaving been manipulated by the hand of man. For example, a polypeptideis considered to be “engineered” when the polypeptide sequencemanipulated by the hand of man. For example, in some embodiments of thepresent invention, an engineered polypeptide comprises a sequence thatincludes one or more amino acid mutations, deletions and/or insertionsthat have been introduced by the hand of man into a referencepolypeptide sequence. Comparably, a cell or organism is considered to be“engineered” if it has been manipulated so that its genetic informationis altered (e.g., new genetic material not previously present has beenintroduced, for example by transformation, mating, somatichybridization, transfection, transduction, or other mechanism, orpreviously present genetic material is altered or removed, for exampleby substitution or deletion mutation, or by mating protocols). As iscommon practice and is understood by those in the art, derivativesand/or progeny of an engineered polypeptide or cell are typically stillreferred to as “engineered” even though the actual manipulation wasperformed on a prior entity.

Epitope: as used herein, includes any moiety that is specificallyrecognized by an immunoglobulin (e.g., antibody or receptor) bindingcomponent. In some embodiments, an epitope is comprised of a pluralityof chemical atoms or groups on an antigen. In some embodiments, suchchemical atoms or groups are surface-exposed when the antigen adopts arelevant three-dimensional conformation. In some embodiments, suchchemical atoms or groups are physically near to each other in space whenthe antigen adopts such a conformation. In some embodiments, at leastsome such chemical atoms are groups are physically separated from oneanother when the antigen adopts an alternative conformation (e.g., islinearized).

Ex vivo: as used herein refers to biologic events that occur outside ofthe context of a multicellular organism. For example, in the context ofcell-based systems, the term may be used to refer to events that occuramong a population of cells (e.g., cell proliferation, cytokinesecretion, etc.) in an artificial environment.

Framework or framework region: as used herein, refers to the sequencesof a variable region minus the CDRs. Because a CDR sequence can bedetermined by different systems, likewise a framework sequence issubject to correspondingly different interpretations. The six CDRsdivide the framework regions on the heavy and light chains into foursub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 ispositioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3between FR3 and FR4. Without specifying the particular sub-regions asFR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FRs within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, FR1, for example, represents thefirst framework region closest to the amino terminal end of the variableregion and 5′ with respect to CDR1, and FRs represents two or more ofthe sub-regions constituting a framework region.

Humanized: as is known in the art, the term “humanized” is commonly usedto refer to antibodies (or antibody components) whose amino acidsequence includes VH and VL region sequences from a reference antibodyraised in a non-human species (e.g., a mouse), but also includesmodifications in those sequences relative to the reference antibodyintended to render them more “human-like”, i.e., more similar to humangermline variable sequences. In some embodiments, a “humanized” antibody(or antibody component) is one that immunospecifically binds to anantigen of interest and that has a framework (FR) region havingsubstantially the amino acid sequence as that of a human antibody, and acomplementary determining region (CDR) having substantially the aminoacid sequence as that of a non-human antibody. A humanized antibodycomprises substantially all of at least one, and typically two, variabledomains (Fab, Fab′, F(ab′)₂, FabC, Fv) in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin(i.e., donor immunoglobulin) and all or substantially all of theframework regions are those of a human immunoglobulin consensussequence. In some embodiments, a humanized antibody also comprises atleast a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin constant region. In some embodiments, ahumanized antibody contains both the light chain as well as at least thevariable domain of a heavy chain. The antibody also may include aC_(H)1, hinge, C_(H)2, C_(H)3, and, optionally, a C_(H)4 region of aheavy chain constant region.

In vitro: The term “in vitro” as used herein refers to events that occurin an artificial environment, e.g., in a test tube or reaction vessel,in cell culture, etc., rather than within a multi-cellular organism.

In vivo: as used herein refers to events that occur within amulti-cellular organism, such as a human and a non-human animal. In thecontext of cell-based systems, the term may be used to refer to eventsthat occur within a living cell (as opposed to, for example, in vitrosystems).

Isolated: as used herein, refers to a substance and/or entity that hasbeen (1) separated from at least some of the components with which itwas associated when initially produced (whether in nature and/or in anexperimental setting), and/or (2) designed, produced, prepared, and/ormanufactured by the hand of man. Isolated substances and/or entities maybe separated from about 10%, about 20%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% of the other components with which they wereinitially associated. In some embodiments, isolated agents are about80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 99%, or more thanabout 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. In some embodiments, as will beunderstood by those skilled in the art, a substance may still beconsidered “isolated” or even “pure”, after having been combined withcertain other components such as, for example, one or more carriers orexcipients (e.g., buffer, solvent, water, etc.); in such embodiments,percent isolation or purity of the substance is calculated withoutincluding such carriers or excipients. To give but one example, in someembodiments, a biological polymer such as a polypeptide orpolynucleotide that occurs in nature is considered to be “isolated”when, a) by virtue of its origin or source of derivation is notassociated with some or all of the components that accompany it in itsnative state in nature; b) it is substantially free of otherpolypeptides or nucleic acids of the same species from the species thatproduces it in nature; c) is expressed by or is otherwise in associationwith components from a cell or other expression system that is not ofthe species that produces it in nature. Thus, for instance, in someembodiments, a polypeptide that is chemically synthesized or issynthesized in a cellular system different from that which produces itin nature is considered to be an “isolated” polypeptide. Alternativelyor additionally, in some embodiments, a polypeptide that has beensubjected to one or more purification techniques may be considered to bean “isolated” polypeptide to the extent that it has been separated fromother components a) with which it is associated in nature; and/or b)with which it was associated when initially produced.

K_(D): as used herein, refers to the dissociation constant of a bindingagent (e.g., an antibody or binding component thereof) from a complexwith its partner (e.g., the epitope to which the antibody or bindingcomponent thereof binds).

Pharmaceutical composition: As used herein, the term “pharmaceuticalcomposition” refers to a composition in which an active agent isformulated together with one or more pharmaceutically acceptablecarriers. In some embodiments, the composition is suitable foradministration to a human or animal subject. In some embodiments, theactive agent is present in unit dose amount appropriate foradministration in a therapeutic regimen that shows a statisticallysignificant probability of achieving a predetermined therapeutic effectwhen administered to a relevant population.

Polypeptide: The term “polypeptide”, as used herein, generally has itsart-recognized meaning of a polymer of at least three amino acids. Thoseof ordinary skill in the art will appreciate that the term “polypeptide”is intended to be sufficiently general as to encompass not onlypolypeptides having a complete sequence recited herein, but also toencompass polypeptides that represent functional fragments (i.e.,fragments retaining at least one activity) of such completepolypeptides. Moreover, those of ordinary skill in the art understandthat protein sequences generally tolerate some substitution withoutdestroying activity. Thus, any polypeptide that retains activity andshares at least about 30-40% overall sequence identity, often greaterthan about 50%, 60%, 70%, or 80%, and further usually including at leastone region of much higher identity, often greater than 90% or even 95%,96%, 97%, 98%, or 99% in one or more highly conserved regions, usuallyencompassing at least 3-4 and often up to 20 or more amino acids, withanother polypeptide of the same class, is encompassed within therelevant term “polypeptide” as used herein. Polypeptides may containL-amino acids, D-amino acids, or both and may contain any of a varietyof amino acid modifications or analogs known in the art. Usefulmodifications include, e.g., terminal acetylation, amidation,methylation, etc. In some embodiments, proteins may comprise naturalamino acids, non-natural amino acids, synthetic amino acids, andcombinations thereof. The term “peptide” is generally used to refer to apolypeptide having a length of less than about 100 amino acids, lessthan about 50 amino acids, less than 20 amino acids, or less than 10amino acids. In some embodiments, proteins are antibodies, antibodyfragments, biologically active portions thereof, and/or characteristicportions thereof.

Prevent or prevention: as used herein when used in connection with theoccurrence of a disease, disorder, and/or condition, refers to reducingthe risk of developing the disease, disorder and/or condition and/or todelaying onset and/or severity of one or more characteristics orsymptoms of the disease, disorder or condition. In some embodiments,prevention is assessed on a population basis such that an agent isconsidered to “prevent” a particular disease, disorder or condition if astatistically significant decrease in the development, frequency, and/orintensity of one or more symptoms of the disease, disorder or conditionis observed in a population susceptible to the disease, disorder, orcondition.

Recombinant: as used herein, is intended to refer to polypeptides thatare designed, engineered, prepared, expressed, created, manufactured,and/or or isolated by recombinant means, such as polypeptides expressedusing a recombinant expression vector transfected into a host cell;polypeptides isolated from a recombinant, combinatorial humanpolypeptide library; polypeptides isolated from an animal (e.g., amouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwisehas been manipulated to express a gene or genes, or gene components thatencode and/or direct expression of the polypeptide or one or morecomponent(s), portion(s), element(s), or domain(s) thereof; and/orpolypeptides prepared, expressed, created or isolated by any other meansthat involves splicing or ligating selected nucleic acid sequenceelements to one another, chemically synthesizing selected sequenceelements, and/or otherwise generating a nucleic acid that encodes and/ordirects expression of the polypeptide or one or more component(s),portion(s), element(s), or domain(s) thereof. In some embodiments, oneor more of such selected sequence elements is found in nature. In someembodiments, one or more of such selected sequence elements is designedin silico. In some embodiments, one or more such selected sequenceelements results from mutagenesis (e.g., in vivo or in vitro) of a knownsequence element, e.g., from a natural or synthetic source such as, forexample, in the germline of a source organism of interest (e.g., of ahuman, a mouse, etc.).

Specific binding: As used herein, the term “specific binding” refers toan ability to discriminate between possible binding partners in theenvironment in which binding is to occur. A binding agent that interactswith one particular target when other potential targets are present issaid to “bind specifically” to the target with which it interacts. Insome embodiments, specific binding is assessed by detecting ordetermining degree of association between the binding agent and itspartner; in some embodiments, specific binding is assessed by detectingor determining degree of dissociation of a binding agent-partnercomplex; in some embodiments, specific binding is assessed by detectingor determining ability of the binding agent to compete an alternativeinteraction between its partner and another entity. In some embodiments,specific binding is assessed by performing such detections ordeterminations across a range of concentrations.

Subject: As used herein, the term “subject” refers an organism,typically a mammal (e.g., a human, in some embodiments includingprenatal human forms). In some embodiments, a subject is suffering froma relevant disease, disorder or condition. In some embodiments, asubject is susceptible to a disease, disorder, or condition. In someembodiments, a subject displays one or more symptoms or characteristicsof a disease, disorder or condition. In some embodiments, a subject doesnot display any symptom or characteristic of a disease, disorder, orcondition. In some embodiments, a subject is someone with one or morefeatures characteristic of susceptibility to or risk of a disease,disorder, or condition. In some embodiments, a subject is a patient. Insome embodiments, a subject is an individual to whom diagnosis and/ortherapy is and/or has been administered.

Therapeutic agent: As used herein, the phrase “therapeutic agent” ingeneral refers to any agent that elicits a desired pharmacologicaleffect when administered to an organism. In some embodiments, an agentis considered to be a therapeutic agent if it demonstrates astatistically significant effect across an appropriate population. Insome embodiments, the appropriate population may be a population ofmodel organisms. In some embodiments, an appropriate population may bedefined by various criteria, such as a certain age group, gender,genetic background, preexisting clinical conditions, etc. In someembodiments, a therapeutic agent is a substance that can be used toalleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduceseverity of, and/or reduce incidence of one or more symptoms or featuresof a disease, disorder, and/or condition. In some embodiments, a“therapeutic agent” is an agent that has been or is required to beapproved by a government agency before it can be marketed foradministration to humans. In some embodiments, a “therapeutic agent” isan agent for which a medical prescription is required for administrationto humans.

Therapeutically Effective Amount: As used herein, the term“therapeutically effective amount” means an amount that is sufficient,when administered to a population suffering from or susceptible to adisease, disorder, and/or condition in accordance with a therapeuticdosing regimen, to treat the disease, disorder, and/or condition. Insome embodiments, a therapeutically effective amount is one that reducesthe incidence and/or severity of, stabilizes one or more characteristicsof, and/or delays onset of, one or more symptoms of the disease,disorder, and/or condition. Those of ordinary skill in the art willappreciate that the term “therapeutically effective amount” does not infact require successful treatment be achieved in a particularindividual. Rather, a therapeutically effective amount may be thatamount that provides a particular desired pharmacological response in asignificant number of subjects when administered to patients in need ofsuch treatment. For example, in some embodiments, term “therapeuticallyeffective amount”, refers to an amount which, when administered to anindividual in need thereof in the context of inventive therapy, willblock, stabilize, attenuate, or reverse a cancer-supportive processoccurring in said individual, or will enhance or increase acancer-suppressive process in said individual. In the context of cancertreatment, a “therapeutically effective amount” is an amount which, whenadministered to an individual diagnosed with a cancer, will prevent,stabilize, inhibit, or reduce the further development of cancer in theindividual. A particularly preferred “therapeutically effective amount”of a composition described herein reverses (in a therapeutic treatment)the development of a malignancy such as a pancreatic carcinoma or helpsachieve or prolong remission of a malignancy. A therapeuticallyeffective amount administered to an individual to treat a cancer in thatindividual may be the same or different from a therapeutically effectiveamount administered to promote remission or inhibit metastasis. As withmost cancer therapies, the therapeutic methods described herein are notto be interpreted as, restricted to, or otherwise limited to a “cure”for cancer; rather the methods of treatment are directed to the use ofthe described compositions to “treat” a cancer, i.e., to effect adesirable or beneficial change in the health of an individual who hascancer. Such benefits are recognized by skilled healthcare providers inthe field of oncology and include, but are not limited to, astabilization of patient condition, a decrease in tumor size (tumorregression), an improvement in vital functions (e.g., improved functionof cancerous tissues or organs), a decrease or inhibition of furthermetastasis, a decrease in opportunistic infections, an increasedsurvivability, a decrease in pain, improved motor function, improvedcognitive function, improved feeling of energy (vitality, decreasedmalaise), improved feeling of well-being, restoration of normalappetite, restoration of healthy weight gain, and combinations thereof.In addition, regression of a particular tumor in an individual (e.g., asthe result of treatments described herein) may also be assessed bytaking samples of cancer cells from the site of a tumor (e.g., over thecourse of treatment) and testing the cancer cells for the level ofmetabolic and signaling markers to monitor the status of the cancercells to verify at the molecular level the regression of the cancercells to a less malignant phenotype. For example, tumor regressioninduced by employing the methods of this invention would be indicated byfinding a decrease in any of the pro-angiogenic markers discussed above,an increase in anti-angiogenic markers described herein, thenormalization (i.e., alteration toward a state found in normalindividuals not suffering from cancer) of metabolic pathways,intercellular signaling pathways, or intracellular signaling pathwaysthat exhibit abnormal activity in individuals diagnosed with cancer.Those of ordinary skill in the art will appreciate that, in someembodiments, a therapeutically effective amount may be formulated and/oradministered in a single dose. In some embodiments, a therapeuticallyeffective amount may be formulated and/or administered in a plurality ofdoses, for example, as part of a dosing regimen.

Variant: As used herein in the context of molecules, e.g., nucleicacids, proteins, or small molecules, the term “variant” refers to amolecule that shows significant structural identity with a referencemolecule but differs structurally from the reference molecule, e.g., inthe presence or absence or in the level of one or more chemical moietiesas compared to the reference entity. In some embodiments, a variant alsodiffers functionally from its reference molecule. In general, whether aparticular molecule is properly considered to be a “variant” of areference molecule is based on its degree of structural identity withthe reference molecule. As will be appreciated by those skilled in theart, any biological or chemical reference molecule has certaincharacteristic structural elements. A variant, by definition, is adistinct molecule that shares one or more such characteristic structuralelements but differs in at least one aspect from the reference molecule.To give but a few examples, a polypeptide may have a characteristicsequence element comprised of a plurality of amino acids havingdesignated positions relative to one another in linear orthree-dimensional space and/or contributing to a particular structuralmotif and/or biological function; a nucleic acid may have acharacteristic sequence element comprised of a plurality of nucleotideresidues having designated positions relative to on another in linear orthree-dimensional space. In some embodiments, a variant polypeptide ornucleic acid may differ from a reference polypeptide or nucleic acid asa result of one or more differences in amino acid or nucleotidesequence. In some embodiments, a variant polypeptide or nucleic acidshows an overall sequence identity with a reference polypeptide ornucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variantpolypeptide or nucleic acid does not share at least one characteristicsequence element with a reference polypeptide or nucleic acid. In someembodiments, a reference polypeptide or nucleic acid has one or morebiological activities. In some embodiments, a variant polypeptide ornucleic acid shares one or more of the biological activities of thereference polypeptide or nucleic acid.

Vector: as used herein, refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors.” Standard techniques may be used forrecombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g., electroporation, lipofection). Enzymatic reactionsand purification techniques may be performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures may be generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. See e.g., Sambrooket al., Molecular Cloning: A Laboratory Manual (2^(nd) ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which isincorporated herein by reference for any purpose.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The present invention relates to anti-AITR antibodies for conversion ofa regulatory T cell into a T_(H)1-like cell. For example, engineeredantibodies provided herein have been modified to enhance antigenaffinity relative to parental antibodies that specifically recognize anepitope within the extracellular domain of human AITR. Specifically, asdescribed herein, the inventors created several exemplary antibodieswith improved affinity for human AITR. Notably, the exemplary anti-AITRantibodies are cable of converting regulatory T cells (T_(reg) cells)into T_(H)1 cells and regulating the quantity of certain T cell cytokinesecretions. Thus the present disclosure provides engineered anti-AITRantibodies with improved properties over reference antibodies, andmoreover demonstrates that these antibodies have surprisingly beneficialactivity in vitro and in vivo.

AITR

The human activation-inducible tumor necrosis factor receptor (AITR) isa member of the TNFR superfamily, and is also known as GITR(glucocorticoid-induced TNFR-related protein), TNFRSF 18 (TNF receptorsuperfamily member 18) or CD357 (Kwon et al., J Biol Chem,274:6056-6061, 1999). AITR is expressed at particularly high levels inT_(reg) cells (regulatory T cells) and activated T cells. AITR refers toany variant, isoform and homolog which is naturally expressed by a celland may specifically mean, but is not limited to, human AITR.Information on AITR is available from a known database such as NCBIGenBank, and an example thereof may include, but is not limited to,NP_004186. Stimulation of AITR generates a unique intracellular signalwhich decreases immunosuppressive function of regulatory T cell andpromotes T_(eff) cell (effector T cell) activity (Shimizu et al., NatImmunol., 3:135-142, 2002). Therefore, in view of immuno-oncology, AITRsignal increases human immunity against tumors and induces cancer celldeath (Sakaguchi, Cell, 101:455-458, 2000).

AITR acts as a trimer rather than a single molecule under physiologicalconditions, and the functional unit of its ligand is a trimer. Accordingto the structural studies on TNFRSF (TNF receptor superfamily) and TNF,three receptor molecules and a TNF trimer—are symmetrically bound andeach receptor fragment is bound to grooves of two adjacent TNF. Thus,the contact interface between TNF and TNFR plays a pivotal role in theTNFR-TNF interaction and structure. This functional significance couldexplain why the amino acid sequence of the contact surface is highlyconserved among species, as well as in the TNFR superfamily (Chan etal., Immunity, 13:419-422, 2000).

In addition, a cell exposed to a specific condition can progress from areceptor trimer to achieve a tetramer of trimers and then generate aprogressive super-signal internally. For this reason, an AITR signal isnot a simple transmission of a single on-off signal, but rather afinely-tuned regulation of signal transduction and it becomes animportant factor in controlling the anti-cancer capacity of T_(reg)cells (Zhou et al., PNAS., 105:5465-5470, 2008). The complexity anddiversity of interactions between TNFR and its ligand, and the numerousavailable epitopes, enables very precise signal regulation. Therefore, amonoclonal antibody (mAb) that can recognize a particular structuralepitope is an optimal candidate for developing a therapeutic agentrelated to AITR. That is, depending upon where the epitope is on theAITR molecule that is recognized by a particular provided anti-AITRantibody, a particular anti-AITR antibody may mimic a natural ligand ofthe AITR to varying degrees, for example, activity may be selectivelymodulated and/or receptor oligomerization may also the adjusted, suchthat the response through the AITR may be modulated in various ways.

Anti-AITR Antibodies and Fragments Thereof

The present disclosure provides, at least in part, engineered anti-AITRantibodies (also described herein as IFN-γ-inducible Regulatory T cellConvertible anti-cancer mAb (IRTCA) and fragments thereof that exhibitmarkedly, and unexpectedly, superior characteristics in vitro and/or invivo. For example, certain provided antibodies have increased affinityrelative to reference anti-AITR antibodies (e.g., anti-AITR antibodiespreviously described in U.S. Pat. Nos. 9,255,151, 9,255,152, and9,309,321, all of which are incorporated herein by reference in theirentirety).

In some embodiments, an IRTCA antibody or antigen-binding antibodyfragment includes 1, 2, or 3 heavy chain CDR sequences that are orinclude a sequence of selected from SEQ ID NO: 8, 9, 10, 14, 15, 16, 17,24, and 25. In some embodiments, an IRTCA antibody or antigen-bindingantibody fragment includes one or more of: a heavy chain CDR1 that is orincludes a sequence selected from SEQ ID NO: 8 and 24, a heavy chainCDR2 that is or includes a sequence selected from SEQ ID NO: 9 and 25and a heavy chain CDR3 that is or includes a sequence selected from SEQID NO: 10, 14, 15, 16, and 17. In some embodiments, an IRTCA antibody orantigen-binding antibody fragment includes each of: a heavy chain CDR1that is or includes a sequence selected from SEQ ID NO: 8 and 24, aheavy chain CDR2 that is or includes a sequence selected from SEQ ID NO:9 and 25 and a heavy chain CDR3 that is or includes a sequence selectedfrom SEQ ID NO: 10, 14, 15, 16, and 17.

In some embodiments, an IRTCA antibody or antigen-binding antibodyfragment includes 1, 2, or 3 light chain CDR sequences that are orinclude a sequence selected from SEQ ID NO: 11, 12, 13, and 18. In someembodiments, an IRTCA antibody or antigen-binding antibody fragmentincludes one or more of: a light chain CDR1 that is or includes asequence of SEQ ID NO: 11, a light chain CDR2 that is or includes asequence of SEQ ID NO: 12, and a light chain CDR3 that is or includes asequence selected from SEQ ID NO: 13 and 18. In some embodiments, anIRTCA antibody or antigen-binding antibody fragment includes each of: alight chain CDR1 that is or includes a sequence of SEQ ID NO: 11, alight chain CDR2 that is or includes a sequence of SEQ ID NO: 12, and alight chain CDR3 that is or includes a sequence selected from SEQ ID NO:13 and 18.

In some embodiments, an IRTCA antibody or antigen-binding fragmentthereof, comprises (a) a heavy chain CDR1 comprising a sequence of SEQID NO: 8 or 24, a heavy chain CDR2 comprising a sequence of SEQ ID NO: 9or 25, and a heavy chain CDR3 comprising at least one sequence selectedfrom SEQ ID NO: 14, 15, 16, and 17; and (b) a light chain CDR1comprising a sequence of SEQ ID NO: 11, a light chain CDR2 comprising asequence of SEQ ID NO: 12 and a light chain CDR3 comprising a sequenceof SEQ ID NO: 13 or 18, wherein the IRTCA antibody or antigen-bindingfragment thereof does not comprise each of a heavy chain CDR1 comprisinga sequence of SEQ ID NO: 8, a heavy chain CDR2 comprising a sequence ofSEQ ID NO: 9, a heavy chain CDR3 comprising a sequence of SEQ ID NO: 10,a light chain CDR1 comprising a sequence of SEQ ID NO: 11, a light chainCDR2 comprising a sequence of SEQ ID NO: 12 and a light chain CDR3comprising a sequence of SEQ ID NO: 13.

In some embodiments, an IRTCA antibody or antigen-binding antibodyfragment includes substantial homology to an antibody or antibodyfragment that includes a heavy chain variable domain that is or includesa sequence selected from SEQ ID NOs: 1, 3, 4, 5, 6, 20, and 21. In someembodiments, an IRTCA antibody or antigen-binding antibody fragmentincludes a heavy chain variable domain that is or includes a sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%,99.3%, 99.4% or 99.5% identical to a sequence selected from SEQ ID NOs:1, 3, 4, 5, 6, 20, and 21. In some embodiments, an IRTCA antibody orantigen-binding antibody fragment includes a heavy chain variable domainthat is or includes a sequence selected from SEQ ID NOs: 1, 3, 4, 5, 6,20, and 21.

In some embodiments, an IRTCA antibody or antigen-binding antibodyfragment includes substantial homology to an antibody or antibodyfragment that includes a light chain variable domain that has orincludes a sequence selected from SEQ ID NOs: 2, 7, 22, and 23. In someembodiments, an IRTCA antibody or antigen-binding antibody fragmentincludes a light chain variable domain that is or includes a sequence atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%,99.3%, 99.4% or 99.5% identical to a sequence selected from SEQ ID NOs:2, 7, 22, and 23. In some embodiments, an IRTCA antibody orantigen-binding antibody fragment includes a light chain variable domainthat is or includes a sequence selected from SEQ ID NOs: 2, 7, 22, and23.

In some embodiments, an IRTCA antibody or antigen-binding antibodyfragment includes substantial homology to an antibody or antibodyfragment that includes a heavy chain variable domain that is or includesa sequence selected from SEQ ID NOs: 1, 3, 4, 5, 6, 20, and 21 and alight chain variable domain that is or includes a sequence selected fromSEQ ID NOs: 2, 7, 22, and 23. In some embodiments, an IRTCA antibody orantigen-binding antibody fragment includes a heavy chain variable domainthat is or includes a sequence at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or 99.5% identical to asequence selected from SEQ ID NOs: 1, 3, 4, 5, 6, 20, and 21 and a lightchain variable domain that is or includes a sequence at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4% or99.5% identical to a sequence selected from SEQ ID NOs: 2, 7, 22, and23. In some embodiments, an IRTCA antibody or antigen-binding antibodyfragment includes a heavy chain variable domain that is or includes asequence selected from SEQ ID NOs: 1, 3, 4, 5, 6, 20, and 21 and a lightchain variable domain that is or includes a sequence selected from SEQID NOs: 2, 7, 22, and 23.

Amino acid sequences of an IRTCA antibody or antigen-binding fragmentbinds of the present disclosure may be substituted through conservativesubstitution. The term “conservative substitution” used herein refers tomodification of a polypeptide in which one or more amino acids aresubstituted with an amino acid having a similar biochemical property soas not to cause the loss of a biological or biochemical function of thecorresponding polypeptide. The term “conservative sequence variant” or“conservative amino acid substitution” used herein is the substitutionof an amino acid residue with an amino acid residue having a similarside chain. Amino acid residues having a similar side chain are definedin the art. Those residues encompass amino acids with a basic side chain(e.g., lysine, arginine, and histidine), amino acids with an acidic sidechain (e.g., aspartic acid and glutamate), amino acids with anon-charged polar side chain (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, and cysteine), amino acids with a non-polarside chain (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, and tryptophan), amino acids with abeta-branched side chain (e.g., threonine, valine, and isoleucine) andamino acids with an aromatic side chain (e.g., tyrosine, phenylalanine,tryptophan, and histidine). Therefore, it is expected that the antibodyof the present invention can have conservative amino acid substitution,and still ensure an activity.

In some embodiments, an IRTCA antibody or antigen-binding fragment ofthe present disclosure may include a constant region selected from anIgG1 constant domain, an IgG2 constant domain, an IgG1/IgG2 hybridconstant domain, a human IgG4 constant domain, an IgA constant domain,an IgE constant domain, an IgM constant domain, and an IgD constantdomain.

In some embodiments, an IRTCA antibody or antigen-binding fragment ofthe present disclosure is or includes an IgA, IgD, IgE, IgM, IgG, orvariants thereof.

In some embodiments, an IRTCA antibody or antigen-binding fragmentincludes a light chain constant region. In some embodiments, an IRTCAantibody or antigen-binding fragment includes a kappa (κ) and/or lambda(λ) light chain and/or a variant thereof.

In some embodiments, an IRTCA antibody or antigen-binding fragment is amonoclonal antibody. In some embodiments, an IRTCA antibody orantigen-binding fragment is a Fab fragment, a Fab′ fragment, a F(ab′)2fragment, a Fv fragment, a disulfide-bonded Fv fragment, a scFvfragment, a single domain antibody, humabody, nanobody, and/or adiabody. In some embodiments, an IRTCA antibody or antigen-bindingfragment is a monovalent antibody. In some embodiments, an IRTCAantibody or antigen-binding fragment is a multivalent antibody. In someembodiments, an IRTCA antibody or antigen-binding fragment is amulti-specific antibody (e.g., a bispecific antibody).

In some embodiments, the present disclosure encompasses methods ofmodifying the carbohydrate content of an antibody of the disclosure byadding or deleting a glycosylation site. Methods for modifying thecarbohydrate content of antibodies are well known in the art andencompassed within the disclosure, see, e.g., U.S. Pat. No. 6,218,149;EP 0 359 096 B1; U.S. Publication No. US 2002/0028486; WO 03/035835;U.S. Publication No. 2003/0115614; U.S. Pat. Nos. 6,218,149; 6,472,511;all of which are incorporated herein by reference in their entirety. Inother embodiments, the present disclosure encompasses methods ofmodifying the carbohydrate content of an antibody of the presentdisclosure by deleting one or more endogenous carbohydrate moieties ofthe antibody.

Engineered glycoforms may be useful for a variety of purposes, includingbut not limited to enhancing or reducing effector function. Engineeredglycoforms may be generated by any method known to one skilled in theart, for example by using engineered or variant expression strains, byco-expression with one or more enzymes, for example DIN-acetylglucosaminyltransferase III (GnTI11), by expressing a moleculecomprising an Fc region in various organisms or cell lines from variousorganisms, or by modifying carbohydrate(s) after the molecule comprisingFc region has been expressed. Methods for generating engineeredglycoforms are known in the art, and include but are not limited tothose described in Umana et al, 1999, Nat. Biotechnol 17:176-180; Davieset al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J BiolChem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473)U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1;PCT WO 02/30954A1; POTILLEGENT™ technology (Biowa, Inc. Princeton,N.J.); GLYCOMAB™ glycosylation engineering technology (GLYCARTbiotechnology AG, Zurich, Switzerland); each of which is incorporatedherein by reference in its entirety. See, e.g., WO 00061739; EA01229125;US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49 each of which isincorporated herein by reference in its entirety.

In some embodiments, an IRTCA antibody or antigen-binding fragment ofthe present disclosure is as an antagonist of human AITR.

In some embodiments, an IRTCA antibody or antigen-binding fragment ofthe present disclosure binds to a human AITR molecule. In someembodiments, an IRTCA antibody or antigen-binding fragment of thepresent disclosure specifically binds to a human AITR molecule.

In some embodiments, an IRTCA antibody or antigen-binding fragment bindsto a sequence that is or includes that of SEQ ID NO: 19. In someembodiments, an IRTCA antibody or antigen-binding fragment binds to anepitope of IRTCA extracellular domain that is or includes a sequence ofSEQ ID NO: 19.

In some embodiments, an IRTCA antibody or antigen-binding fragment ofthe present disclosure binds to an AITR molecule with a binding affinity(K_(D)) of 1×10⁻⁷ to 1×10⁻¹² M. In some embodiments, an IRTCA antibodyor antigen-binding fragment of the present disclosure binds to a humanAITR molecule with a binding affinity (K_(D)) of 1×10⁻⁸ to 1×10⁻¹² M.Binding affinity (K_(D)) may be measured, for example, by surfaceplasmon resonance, for example, using a BIACORE system.

In some embodiments, an IRTCA antibody or antigen-binding fragment ofthe present disclosure binds to a human AITR molecule or a fragmentthereof at a binding affinity (K_(D)) of less than 1.0×10⁻⁸ M. In someembodiments, an IRTCA antibody or antigen-binding fragment of thepresent disclosure binds to a human AITR molecule or a fragment thereofat a binding affinity (K_(D)) of less than 1.0×10⁻⁹ M. In someembodiments, an IRTCA antibody or antigen-binding fragment of thepresent disclosure binds to a human AITR molecule or a fragment thereofat a binding affinity (K_(D)) of less than 1.0×10⁻¹⁰ M.

In some embodiments, an IRTCA antibody or antigen-binding fragment ofthe present disclosure fails to bind or weakly binds a non-primate AITRpolypeptide (e.g., a canine, mouse and rat AITR polypeptide). In someembodiments, an IRTCA antibody or antigen-binding fragment of thepresent disclosure binds efficiently to human or monkey AITR. Thisbinding affinity suggests that the structure and/or sequence of epitopefor a primate AITR antibody may be quite different from canine, mouseand rat.

In some embodiments, an IRTCA antibody or antigen-binding fragment ofthe present disclosure binds CD4⁺ T cells expressing human AITR. In someembodiments, an IRTCA antibody or antigen-binding fragment of thepresent disclosure affects T cell populations. In some embodiments, anIRTCA antibody or antigen-binding fragment of the present disclosureconverts regulatory T cells (nT_(reg) cells) to T_(H)1-like(IFN-γ-positive) cells. In some embodiments, an IRTCA antibody orantigen-binding fragment of the present disclosure converts inducibleregulatory T cells (iT_(reg) cells) to T_(H)1-like (IFN-γ-positive)cells. In some embodiments, an IRTCA antibody or antigen-bindingfragment of the present disclosure converts effector T cells (T_(eff)cells) to T_(H)1 (IFN-γ-positive) cells. In some embodiments, an IRTCAantibody or antigen-binding fragment of the present disclosure decreasesa population of regulatory T cells (T_(reg) cells). In some embodiments,an IRTCA antibody or antigen-binding fragment of the present disclosurecauses a population of cells to decrease in secretion of a T_(reg)cytokine (e.g., TGF-β).

In some embodiments, an IRTCA antibody or antigen-binding fragment ofthe present disclosure is characterized by low toxicity (e.g., a lowdegree of post administration cell death). In some embodiments, an IRTCAantibody or antigen-binding fragment of the present disclosure ischaracterized by low hepatoxicity. In some embodiments, a subject thathas been administered an IRTCA antibody or antigen-binding fragment ofthe present disclosure at a therapeutic dose has levels of one or moreof ALT, AST and total bilirubin in a normal range. In some embodiments,an IRTCA antibody or antigen-binding fragment of the present disclosureis characterized by an ability to treat patients for extended periodswith measurable alleviation of symptoms and low and/or acceptabletoxicity. Low or acceptable immunogenicity and/or high affinity, as wellas other suitable properties, can contribute to the therapeutic resultsachieved. “Low immunogenicity” is defined herein as raising significantHAHA, HACA or HAMA responses in less than about 75%, or preferably lessthan about 50% of the patients treated and/or raising low titers in thepatient treated (Elliott et al., Lancet 344:1125-1127 (1994), entirelyincorporated herein by reference).

In some embodiments, an IRTCA antibody or antigen-binding fragment ofthe present disclosure is characterized by its ability to bind to atleast one, at least two, at least three, at least four, or at least fiveresidues of SEQ ID NO: 19 within the extracellular domain of human AITRpeptide. In some embodiments, an IRTCA antibody or antigen-bindingfragment that bind to at least one, at least two, at least three, atleast four, or at least five residues of SEQ ID NO: 19 binds to humanAITR with improved affinity as compared to a reference antibody orantigen-binding fragment (e.g, as compared to IRTCA-A). In someembodiments, an IRTCA antibody or antigen-binding fragment that bind toat least one, at least two, at least three, at least four, or at leastfive residues of SEQ ID NO: 19 binds to human AITR with a bindingaffinity (K_(D)) of between 1×10⁻⁷ to 1×10⁻¹² M.

In some embodiments, an IRTCA antibody or antigen-binding fragment ofthe present disclosure is characterized by its ability to bind to atleast one, at least two, at least three, at least four, or all fiveresidues of SEQ ID NO: 27 within the extracellular domain of human AITRpeptide. In some embodiments, an IRTCA antibody or antigen-bindingfragment that bind to at least one, at least two, at least three, atleast four, or all five residues of SEQ ID NO: 27 binds to human AITRwith improved affinity as compared to a reference antibody orantigen-binding fragment (e.g, as compared to IRTCA-A). In someembodiments, an IRTCA antibody or antigen-binding fragment that bind toat least one, at least two, at least three, at least four, or all fiveresidues of SEQ ID NO: 27 binds to human AITR with a binding affinity(K_(D)) of between 1×10⁻⁷ to 1×10⁻¹² M.

In other embodiments, an IRTCA antibody or antigen-binding fragment ofthe present disclosure is characterized by its ability to bind to atleast one, at least two, at least three, at least four, or at least fiveresidues of SEQ ID NO: 28 or 30 within the extracellular domain of humanAITR peptide. In some embodiments, an IRTCA antibody or antigen-bindingfragment that bind to at least one, at least two, at least three, atleast four, or at least five residues of SEQ ID NO: 28 or 30 binds tohuman AITR with improved affinity as compared to a reference antibody orantigen-binding fragment (e.g, as compared to IRTCA-A). In someembodiments, an IRTCA antibody or antigen-binding fragment that bind toat least one, at least two, at least three, at least four, or at leastfive residues of SEQ ID NO: 28 or 30 binds to human AITR with a bindingaffinity (K_(D)) of between 1×10⁻⁷ to 1×10⁻¹² M.

In some embodiments, an IRTCA antibody or antigen-binding fragment ofthe present disclosure is characterized by its ability to bind to atleast one, at least two, at least three, at least four, or all fiveresidues of SEQ ID NO: 29 or 31 within the extracellular domain of humanAITR peptide. In some embodiments, an IRTCA antibody or antigen-bindingfragment that bind to at least one, at least two, at least three, atleast four, or all five residues of SEQ ID NO: 29 or 31 binds to humanAITR with improved affinity as compared to a reference antibody orantigen-binding fragment (e.g, as compared to IRTCA-A). In someembodiments, an IRTCA antibody or antigen-binding fragment that bind toat least one, at least two, at least three, at least four, or all fiveresidues of SEQ ID NO: 29 or 31 binds to human AITR with a bindingaffinity (K_(D)) of between 1×10⁻⁷ to 1×10⁻¹² M.

Nucleic Acids

The disclosure provides polynucleotides comprising a nucleotide sequenceencoding IRTCA antibodies of the present disclosure and fragmentsthereof. IRTCA antibodies and fragments thereof as described herein maybe produced from nucleic acid molecules using molecular biologicalmethods known to the art. Nucleic acids of the present disclosureinclude, for example, DNA and/or RNA.

In some embodiments, nucleic acid constructs include regions that encodean IRTCA antibody or fragment thereof (e.g., H1F1, H1F1M69, H1F1M74). Insome embodiments, such antibodies or fragments thereof will includeV_(H) and/or V_(L) regions. An IRTCA antibody or fragment thereof may beidentified and/or selected for a desired binding and/or functionalproperties, and variable regions of said antibody isolated, amplified,cloned and/or sequenced. Modifications may be made to the V_(H) andV_(L) nucleotide sequences, including additions of nucleotide sequencesencoding amino acids and/or carrying restriction sites, and/orsubstitutions of nucleotide sequences encoding amino acids. In someembodiments, a nucleic acid sequence may or may not include an intronsequence.

Where appropriate, nucleic acid sequences that encode IRTCA antibodiesand fragments thereof (e.g., H1F1, H1F1M69, H1F1M74) may be modified toinclude codons that are optimized for expression in a particular celltype or organism (e.g., see U.S. Pat. Nos. 5,670,356 and 5,874,304).Codon optimized sequences are synthetic sequences, and preferably encodethe identical polypeptide (or a biologically active fragment of a fulllength polypeptide which has substantially the same activity as the fulllength polypeptide) encoded by the non-codon optimized parentpolynucleotide. In some embodiments, the coding region of the geneticmaterial encoding antibody components, in whole or in part, may includean altered sequence to optimize codon usage for a particular cell type(e.g., a eukaryotic or prokaryotic cell). For example, a coding sequencefor a humanized heavy (or light) chain variable region as describedherein may be optimized for expression in a bacterial cells.Alternatively, the coding sequence may be optimized for expression in amammalian cell (e.g., a CHO cell). Such a sequence may be described as acodon-optimized sequence.

Nucleic acid constructs of the present disclosure may be inserted intoan expression vector or viral vector by methods known to the art, andnucleic acid molecules may be operably linked to an expression controlsequence. A vector comprising any of the above-described nucleic acidmolecules, or fragments thereof, is further provided by the presentdisclosure. Any of the above nucleic acid molecules, or fragmentsthereof, can be cloned into any suitable vector and can be used totransform or transfect any suitable host. The selection of vectors andmethods to construct them are commonly known to persons of ordinaryskill in the art and are described in general technical references (see,in general, “Recombinant DNA Part D,” Methods in Enzymology, Vol. 153,Wu and Grossman, eds., Academic Press (1987)).

In some embodiments, conventionally used techniques, such as, forexample, electrophoresis, calcium phosphate precipitation, DEAE-dextrantransfection, lipofection, etc. may be used to introduce a foreignnucleic acid (DNA or RNA) into a prokaryotic or eukaryotic host cell.Desirably, a vector may include regulatory sequences, such astranscription and translation initiation and termination codons, whichare specific to the type of host (e.g., bacterium, fungus, plant oranimal) into which the vector is to be introduced, as appropriate andtaking into consideration whether the vector is DNA or RNA. In someembodiments, a vector comprises regulatory sequences that are specificto the genus of the host. Preferably, a vector comprises regulatorysequences that are specific to the species of the host.

In addition to the replication system and the inserted nucleic acid, anucleic acid construct can include one or more marker genes, which allowfor selection of transformed or transfected hosts. Marker genes includebiocide resistance, e.g., resistance to antibiotics, heavy metals, etc.,complementation in an auxotrophic host to provide prototrophy, and thelike.

Suitable vectors include those designed for propagation and expansion orfor expression or both. For example, a cloning vector is selected fromthe group consisting of the pUC series, the pBluescript series(Stratagene, La Jolla, Calif.), the pET series (Novagen, Madison, Wis.),the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λ6T10,λ6T11, λZapII, (Stratagene), λEMBL4, and πNM1149, also can be used.Examples of plant expression vectors include pBI110, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-C1, pMAM and pMAMneo (Clontech). The TOPO cloning system(Invitrogen, Carlsbad, Calif.) also can be used in accordance with themanufacturer's recommendations.

An expression vector can comprise a native or nonnative promoteroperably linked to an isolated or purified nucleic acid molecule asdescribed above. Selection of promoters, e.g., strong, weak, inducible,tissue-specific and developmental-specific, is within the skill in theart. Similarly, combining of a nucleic acid molecule, or fragmentthereof, as described above with a promoter is also within the skill inthe art.

Suitable viral vectors include, for example, retroviral vectors,parvovirus-based vectors, e.g., adeno-associated virus (AAV)-basedvectors, AAV-adenoviral chimeric vectors, and adenovirus-based vectors,and lentiviral vectors, such as Herpes simplex (HSV)-based vectors.These viral vectors can be prepared using standard recombinant DNAtechniques described in, for example, Sambrook et al., MolecularCloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989); and Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates and John Wiley & Sons,New York, N.Y. (1994).

A retroviral vector is derived from a retrovirus. Retrovirus is an RNAvirus capable of infecting a wide variety of host cells. Upon infection,the retroviral genome integrates into the genome of its host cell and isreplicated along with host cell DNA, thereby constantly producing viralRNA and any nucleic acid sequence incorporated into the retroviralgenome. As such, long-term expression of a therapeutic factor(s) isachievable when using retrovirus. Retroviruses contemplated for use ingene therapy are relatively non-pathogenic, although pathogenicretroviruses exist. When employing pathogenic retroviruses, e.g., humanimmunodeficiency virus (HIV) or human T-cell lymphotrophic viruses(HTLV), care must be taken in altering the viral genome to eliminatetoxicity to the host. A retroviral vector additionally can bemanipulated to render the virus replication-deficient. As such,retroviral vectors are considered particularly useful for stable genetransfer in vivo. Lentiviral vectors, such as HIV-based vectors, areexemplary of retroviral vectors used for gene delivery. Unlike otherretroviruses, HIV-based vectors are known to incorporate their passengergenes into non-dividing cells and, therefore, can be of use in treatingpersistent forms of disease.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra).

In some embodiments, nucleic acids and vectors of the present disclosuremay be isolated and/or purified. The present disclosure also provides acomposition comprising an above-described isolated or purified nucleicacid molecule, optionally in the form of a vector. Isolated nucleicacids and vectors may be prepared using standard techniques known in theart including, for example, alkali/SDS treatment, CsCl binding, columnchromatography, agarose gel electrophoresis and other techniques wellknown in the art. The composition can comprise other components asdescribed further herein.

In some embodiments, nucleic acid molecules are inserted into a vectorthat is able to express an IRTCA antibody or fragment thereof whenintroduced into an appropriate host cell. Appropriate host cellsinclude, but are not limited to, bacterial, yeast, insect, and mammaliancells. Exemplary host cells include prokaryotes (e.g., E. coli) andeukaryotes (e.g., a COS or a CHO cell). Mammalian host cells that couldbe used include human HeLa, HEK293, H9 and Jurkat cells, mouse NIH3T3and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cellsand Chinese hamster ovary (CHO) cells (e.g., DG44 cells). In someembodiments, a mammalian host cell suitable for the expression of theantibody may be a Chinese Hamster Ovary (CHO) cell (for example,including DHFR-CHO cells used along with a DHFR-selectable marker), anNSO myeloma cell, a COS cell or an SP2 cell. In some embodiments thehost cell is selected from the group consisting of E. coli, P. pastoris,Sf9, COS, HEK293, Expi293, CHO-S, CHO-DG44, CHO-K1 and a mammalianlymphocyte.

Any method(s) known to one skilled in the art for the insertion of DNAfragments into a vector may be used to construct expression vectorsencoding an IRTCA antibody or fragment thereof of the present disclosureunder control of transcriptional/translational control signals. Thesemethods may include in vitro recombinant DNA and synthetic techniquesand in vivo recombination (See, e.g., Ausubel, supra; or Sambrook,supra).

Production of Antibodies

Antibodies and antigen-binding fragments of the present invention may beprepared and/or purified by any technique known in the art, which allowsfor the subsequent formation of a stable antibody or antibody fragment.

A nucleic acid encoding IRTCA antibody and/or antigen-binding fragmentof the present disclosure may be easily isolated and sequenced byconventional procedures. For example, an oligonucleotide primer designedto specifically amplify corresponding heavy chain and light chain-codingregions from a hybridoma or phage template DNA may be used. Isolatednucleic acids may be inserted into an expression vector, and thendesired monoclonal antibodies may be produced from a suitable host cell(that is, transformant) transformed by introducing the expression vectorto the host cell. In some embodiments, a method for preparing an IRTCAantibody and/or antigen-binding fragment of the present disclosure mayinclude amplifying an expression vector including a nucleic acidencoding the antibody, but is not limited thereto.

In some embodiments, a host cell is eukaryotic host cell, including, forexample, yeast, higher plant, insect and mammalian cells. Depending uponthe host employed in a recombinant production procedure, antibodies andantibody fragments of the present disclosure can be glycosylated or canbe non-glycosylated. In some embodiments, a recombinant expressionvector encoding an IRTCA antibody and/or antigen-binding fragment of thepresent disclosure is introduced into a mammalian host cell and anantibody may be prepared by culturing the host cell for a sufficienttime to express the antibody. In some embodiments, a mammalian host cellis cultured for a sufficient time to secrete an antibody or antibodyfragment of the present disclosure in a culture medium.

In some embodiments, an expressed antibody of the present disclosure maybe uniformly purified after being isolated from the host cell. Isolationand/or purification of an antibody of the present disclosure may beperformed by a conventional method for isolating and purifying aprotein. For example, not wishing to be bound by theory, an IRTCAantibody and/or antigen-binding fragment of the present disclosure canbe recovered and purified from recombinant cell cultures by well-knownmethods including, but not limited to, protein A purification, protein Gpurification, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,NY, N.Y., (1997-2001), e.g., chapters 1, 4, 6, 8, 9, and 10, eachentirely incorporated herein by reference. In some embodiments, anantibody of the present disclosure may be isolated and/or purified byadditionally combining filtration, superfiltration, salting out,dialysis, etc.

Purified IRTCA antibodies and/or antigen-binding fragments of thepresent disclosure can be characterized by, for example, ELISA, ELISPOT,flow cytometry, immunocytology, BIACORE™ analysis, SAPIDYNE KINEXA™kinetic exclusion assay, SDS-PAGE and Western blot, or by HPLC analysisas well as by a number of other functional assays disclosed herein.

Therapeutic Applications

The present disclosure encompasses a recognition that engineeredantibodies and antigen-binding fragments may be useful for diagnosis,prevention, and/or treatment of certain diseases such as, for example,cancer. Any of the IRTCA antibodies or antigen-binding fragmentsprovided herein may be used in therapeutic methods. For example, anIRTCA antibody or antigen-binding fragment of the present disclosure canbe used as immunotherapeutic agents, for example in the treatment of amalignant disease (e.g., cancer).

The present disclosure provides methods for treating and/or preventing amalignant disease, said methods including administering an IRTCAantibody or antigen-binding fragment of the present disclosure to asubject. Methods for modulating or treating at least one malignantdisease in a cell, tissue, organ, animal or patient, include, but arenot limited to, cancer.

Cancer treatments in the context of the present disclosure may bemediated through increasing T_(H)1-like cells and associated cytokines(e.g., IFN-γ). T_(H)1 cells that secrete IFN-γ are known to mediateimmune responses to intracellular pathogens and to prevent tumor-relatedcancers. Inflammation caused by T_(H)1 cells in tumors is known to notstimulate, but rather to prevent cancers (Haabeth O A et al., NatCommun, 2:240, 2011). In tumors, T_(reg) cells promotes local tumorgrowth. Therefore, it is considered to be an important part for cancertreatment to down-regulate regulatory T cells in a tumor (Liu Z et al.,J Immunol, 182(10): 6160-7, 2009). Therefore, conversion of T_(reg)cells into T_(H)1 cells can be very useful prevention or treatment ofcancers.

The conversion of Treg cells into T_(H)1 cells is related to thesignaling pathway and transcription factors related to T_(H)1differentiation. In particular, the Foxp3 transcription factor is aspecific intracellular marker of T_(reg) cells, and if T_(reg) cells aredifferentiated to T_(H)1 cells, the intracellular level of Foxp3 isdownregulated.

Specifically, it has been demonstrated that binding of an IRTCA antibodyor antigen-binding fragment of the present disclosure to human AITRincreases conversion of regulatory T cells (nT_(reg) cells), inducibleregulatory T cells (iT_(reg) cells), or effector T cells (T_(eff) cells)to T_(H)1-like (IFN-γ-positive) cells. In some embodiments, therapeutictreatment with an IRTCA antibody or antigen-binding fragment of thepresent disclosure can reduce and/or inhibit growth of cancer cells.

In some embodiments, the present disclosure provides a method fordelaying or inhibiting tumor growth, comprising regulation of cytokinesecretion in vivo or in vitro by administering an IRTCA antibody orantigen-binding fragment of the present disclosure. In some embodiments,the present disclosure provides a method for reducing tumor burden,comprising regulation of cytokine secretion in vivo or in vitro byadministering an IRTCA antibody or antigen-binding fragment of thepresent disclosure.

In some embodiments, the present disclosure provides a method fortreating cancer or tumor by monitoring to a biological subject of canceror tumor to be treated, comprising: (i) administrating an IRTCA antibodyor antigen-binding fragment of the present disclosure to a subject, (ii)separating then isolating a biological sample from the subject, (iii)measuring a secretion amount of INF-γ or TGF-β from the sample andestimating a proportion ratio and (iv) determining a therapeuticallyeffective amount of the antibody or antigen-binding fragment thereof bycomparing the control samples which are administrated or notadministrated with the IRTCA antibody or antigen-binding fragmentthereof.

In some embodiments, the present disclosure provides a method oftreating a subject in need thereof, the method comprising a step ofadministering to the subject a composition that comprises or delivers anIRTCA antibody or antigen-binding fragment of the present disclosureand/or a nucleic acid the same. In some embodiments, a subject has or isat risk for developing cancer. In some embodiments, the presentdisclosure provides a method for preventing or treating cancer or tumorof a patient, which includes administering a therapeutically effectiveamount of the IRTCA antibody or the antigen-binding fragment thereof toa patient with cancer or tumor.

In some embodiments, the present disclosure provides a method ofinducing an immune response in a subject in need thereof, the methodcomprising a step of administering to the subject a composition thatcomprises or delivers an IRTCA antibody or antigen-binding fragment ofthe present disclosure and/or a nucleic acid the same. In someembodiments, a subject has or is at risk for developing cancer.

In some embodiments, the present disclosure provides a method ofenhancing an immune response or increasing the activity of an immunecell in a subject in need thereof, the method comprising a step ofadministering to the subject a composition that comprises or delivers anIRTCA antibody or antigen-binding fragment of the present disclosureand/or a nucleic acid the same. In some embodiments, a subject has or isat risk for developing cancer.

Cancers suitable for treatment with method of the present disclosure caninclude, but are not limited to, bladder cancer, breast cancer, cervicalcancer, colon cancer, endometrial cancer, esophageal cancer, fallopiantube cancer, gall bladder cancer, gastrointestinal cancer, head and neckcancer, hematological cancer, laryngeal cancer, liver cancer, lungcancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primaryperitoneal cancer, salivary gland cancer, sarcoma, stomach cancer,thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma,and prostate cancer. In some embodiments, a cancer for treatment with anIRTCA antibody or antigen-binding fragment of the present disclosure mayinclude, but is not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphomas), blastoma, sarcoma and leukemia. In someembodiments, cancer may include squamous cell carcinoma, small cell lungcancer, non-small cell lung cancer, lung adenocarcinoma, squamous cellcarcinoma of the lung, peritoneal cancer, hepatocellular carcinoma,gastric cancer, pancreatic cancer, glioma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatocellular carcinoma, breastcancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary carcinoma, kidney cancer, prostate cancer, vulvarcancer, thyroid cancer, liver carcinoma, leukemia and otherlymphoproliferative disorders, and various types of head and neckcancer.

A composition including an IRTCA antibody or antigen-binding fragment ofthe present disclosure may be administered at a pharmaceuticallyeffective amount to treat cancer cells or metastasis thereof, or inhibitthe growth of cancer. For use in therapeutic methods, an IRTCA antibodyor antigen-binding fragment of the present disclosure would beformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the age of thepatient, the weight of the patient, the cause of the disorder, the siteof delivery of the agent, the method of administration, the schedulingof administration, and other factors known to medical practitioners.

The present disclosure provides high affinity IRTCA antibodies that mayhave superior properties relative to a reference antibody. The presentdisclosure encompasses a recognition that these antibodies may haveimproved ability to induce T cell conversion and/or secretion ofcytokines such as IFN-γ. Accordingly, the present disclosure encompassesa recognition that an IRTCA antibody or antigen binding fragment of thepresent disclosure may be administered a dose lower than referenceantibody.

In some embodiments composition that includes an IRTCA antibody orantigen-binding fragment of the present disclosure may be administeredto a patient as a bolus or by continuous injection when needed. In someembodiments, bolus administration is of an IRTCA Fab of the presentdisclosure and may be administered at a dose of 0.0025 to 100 mg/kg,0.025 to 0.25 mg/kg, 0.010 to 0.10 mg/kg, or 0.10 to 0.50 mg/kg. In thecase of the continuous injection, the antibody of the present inventionpresented as a Fab fragment may be administered at a dose of 0.001 to100 mg/kg/min, 0.0125 to 1.25 mg/kg/min, 0.010 to 0.75 mg/kg/min, 0.010to 1.0 mg/kg/min or 0.10 to 0.50 mg/kg/min for 1 to 24 hours, 1 to 12hours, 2 to 12 hours, 6 to 12 hours, 2 to 8 hours, or 1 to 2 hours. Insome embodiment, an antibody of the present disclosure is or comprises afull-length antibody (having a complete constant domain). In someembodiments, a full-length antibody is administered at a dose ofapproximately 0.01 to 10 mg/kg, 1 to 8 mg/kg, or 2 to 6 mg/kg. In someembodiments, a full-length antibody is administered by injection for 30to 35 minutes. Administration frequency may vary depending on theseverity of a condition. For example, the frequency may be once every 2to 7 days, once a week, or once every 1, 2, 3 or 4 weeks.

In some embodiments, a composition may be administered to a patient bysubcutaneous injection. Specifically, the antibody may be administeredto a patient at a dose of 0.1 to 100 mg by subcutaneous injection onceevery 2 to 7 days, every week, once every two weeks, or every month.

A composition including an IRTCA antibody or antigen-binding fragment ofthe present disclosure may be administered to a subject who has beenadministered or will be administered one or more additional anticancertherapies selected from ionizing radiation, a chemotherapeutic agent, anantibody agent, and a cell-based therapy, such that the subject receivestreatment with both. In some embodiments, the one or more additionalanticancer therapies comprise an immune checkpoint inhibitor, IL-12,GM-CSF, an anti-CD4 agent, cisplatin, fluorouracil, doxorubicin,irinotecan, paclitaxel, indoleamine 2,3-dioxygenase-1 (IDO1) inhibitoror cyclophosphamide.

Various embodiments of the present invention may also be useful fordetermining an advantageous dosing regimen for one or more providedIRTCA antibodies and/or antigen binding fragments thereof, nucleic acidmolecules, recombinant vectors, and/or host cells. For example, in someembodiments, the present invention provides methods of determining adose of an IRTCA antibody or antigen binding fragment thereof fortherapeutic treatment of a subject in need thereof, the method includingthe steps of: (a) providing or obtaining a measurement of secreted IFN-γin a biological sample from the subject, wherein the subject has beenadministered a composition that comprises or delivers an amount of aprovided IRTCA antibody or antigen-binding fragment thereof, and (b)comparing the measurement of secreted IFN-γ to a reference value,wherein if the measurement of secreted IFN-γ is higher or lower than thereference value, adjusting the amount of the IRTCA antibody or antigenbinding fragment thereof to be administered, thereby determining a dosefor therapeutic treatment of a subject.

In some embodiments, a reference value may be a level of IFN-γ in abiological sample from the subject prior to administration of theprovided IRTCA antibody or antigen-binding fragment thereof. In someembodiments, if a measured amount of secreted IFN-γ in a biologicalsample from the subject is higher than a reference value, then treatmentis maintained at substantially the same level given previously. In someembodiments, if a measured amount of secreted IFN-γ in a biologicalsample from the subject is higher than a reference value, then the nexttreatment is given at a lower dose than the previous treatment. In someembodiments, if a measured amount of secreted IFN-γ in a biologicalsample from the subject is higher than a reference value, then treatmentis ceased for a period of time. In some embodiments, if a measuredamount of secreted IFN-γ in a biological sample from the subject issubstantially the same as or lower than a reference value, then the nexttreatment is given at a higher dose than the previous treatment.

By way of additional example, in some embodiments, the present inventionprovides methods of determining a dose of an IRTCA antibody or antigenbinding fragment thereof for therapeutic treatment of a subject in needthereof, including the steps of: (a) providing or obtaining ameasurement of T_(reg) cell population in a biological sample from thesubject, wherein the subject has been administered a composition thatcomprises or delivers an amount of a provided IRTCA antibody orantigen-binding fragment thereof; and (b) comparing the measurement ofT_(reg) cell population to a reference value, wherein if the measurementof T_(reg) cell population is higher or lower than the reference value,adjusting the amount of the IRTCA antibody or antigen binding fragmentthereof to be administered, thereby determining a dose for therapeutictreatment of a subject.

In some embodiments, a reference value may be a level of Treg cellpopulation in a biological sample from the subject prior toadministration of the provided IRTCA antibody or antigen-bindingfragment thereof. In some embodiments, if a measured amount of T_(reg)cell population in a biological sample from the subject is lower than areference value, then treatment is maintained at substantially the samelevel given previously. In some embodiments, if a measured amount ofT_(reg) cell population in a biological sample from the subject is lowerthan a reference value, then the next treatment is given at a lower dosethan the previous treatment. In some embodiments, if a measured amountof T_(reg) cell population in a biological sample from the subject islower than a reference value, then treatment is ceased for a period oftime. In some embodiments, if a measured amount of T_(reg) cellpopulation in a biological sample from the subject is substantially thesame as or higher than a reference value, then the next treatment isgiven at a higher dose than the previous treatment.

By way of additional example, in some embodiments, the present inventionprovides methods of determining a dose of an IRTCA antibody or antigenbinding fragment thereof for therapeutic treatment of a subject in needthereof, including the steps of: (a) providing or obtaining ameasurement of IFN-γ-secreting T cell population in a biological samplefrom the subject, wherein the subject has been administered acomposition that comprises or delivers an amount of a provided IRTCAantibody or antigen-binding fragment thereof; (b) providing or obtaininga measurement of T_(reg) cell population in a biological sample from thesubject, wherein the subject has been administered a composition thatcomprises or delivers an amount of a provided IRTCA antibody orantigen-binding fragment thereof; (c) calculating the ratio of themeasurement of IFN-γ-secreting T cell population to the measurement ofT_(reg) cell population; and (d) comparing the ratio to a referencevalue, wherein if ratio is higher or lower than the reference value,adjusting the amount of the IRTCA antibody or antigen binding fragmentthereof to be administered, thereby determining a dose for therapeutictreatment of a subject.

In some embodiments, a reference value may be a ratio of a level ofIFN-γ-secreting T cell population in a biological sample form a subjectto a level of T_(reg) cell population in the same biological sample fromthe subject prior to administration of the provided IRTCA antibody orantigen-binding fragment thereof. In some embodiments, if a calculatedratio in a biological sample from the subject is higher than a referencevalue, then treatment is maintained at the same level given previously.In some embodiments, if a calculated ratio in a biological sample fromthe subject is higher than a reference value, then the next treatment isgiven at a lower dose than the previous treatment. In some embodiments,if a calculated ratio in a biological sample from the subject is higherthan a reference value, then treatment is ceased for a period of time.In some embodiments, if a calculated ratio in a biological sample fromthe subject is substantially the same as or lower than a referencevalue, then the next treatment is given at a higher dose than theprevious treatment.

Compositions

Provided herein, in some embodiments, are compositions comprisingantibodies and antigen binding fragments that specifically bind to anepitope of an AITR polypeptide. Compositions of the present disclosure(e.g., compositions that deliver an IRTCA antibody or antibody fragment)may include any suitable and effective amount of a composition for usein delivering a provided IRTCA antibody or antibody fragment to a cell,tissue, organ, animal or patient in need of such modulation, treatmentor therapy. Also provided herein are compositions that include convertedcell populations (e.g., to T_(H)1-like cells) that have been generatedvia a method of the present disclosure (e.g., a method that includes astep contacting a cell with an IRTCA antibody or antibody fragment).

Compositions of the present disclosure include pharmaceuticalcompositions that include an IRTCA antibody or antigen-binding fragmentdisclosed herein and/or a cell population obtained by a method disclosedherein. In some embodiments, a pharmaceutical composition can include abuffer, a diluent, an excipient, or any combination thereof. In someembodiments, a composition, if desired, can also contain one or moreadditional therapeutically active substances.

In some embodiments, an IRTCA antibody, antigen-binding fragment and/orcell population of the present disclosure are suitable foradministration to a mammal (e.g., a human). Although the descriptions ofpharmaceutical compositions provided herein are principally directed topharmaceutical compositions that are suitable for ethical administrationto humans, it will be understood by the skilled artisan that suchcompositions are generally suitable for administration to animals of allsorts. Modification of pharmaceutical compositions suitable foradministration to humans in order to render the compositions suitablefor administration to various animals is well understood, and theordinarily skilled veterinary pharmacologist can design and/or performsuch modification with merely ordinary, if any, experimentation.

In some embodiments, provided compositions may be formulated forparenteral administration. For example, a pharmaceutical compositionprovided herein may be provided in a sterile injectable form (e.g., aform that is suitable for subcutaneous injection or intravenousinfusion). For example, in some embodiments, a pharmaceuticalcompositions is provided in a liquid dosage form that is suitable forinjection. In some embodiments, a pharmaceutical composition is providedas powders (e.g., lyophilized and/or sterilized), optionally undervacuum, which can be reconstituted with an aqueous diluent (e.g., water,buffer, salt solution, etc.) prior to injection. In some embodiments, apharmaceutical composition is diluted and/or reconstituted in water,sodium chloride solution, sodium acetate solution, benzyl alcoholsolution, phosphate buffered saline, etc. In some embodiments, a powdershould be mixed gently with the aqueous diluent (e.g., not shaken).

In some embodiments, an IRTCA antibody, antigen-binding fragment, and/orcell population of the present disclosure is formulated with apharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, dextrose solution, and1-10% human serum albumin. Liposomes and non-aqueous vehicles such asfixed oils can also be used. A vehicle or lyophilized powder can containadditives that maintain isotonicity (e.g., sodium chloride, mannitol)and chemical stability (e.g., buffers and preservatives). In someembodiments, a formulation is sterilized by known or suitabletechniques.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a diluent oranother excipient and/or one or more other accessory ingredients, andthen, if necessary and/or desirable, shaping and/or packaging theproduct into a desired single- or multi-dose unit.

In some embodiments, a pharmaceutical composition including an IRTCAantibody, antigen-binding fragment, and/or cell population of thepresent disclosure can be included in a container for storage oradministration, for example, an vial, a syringe (e.g., an IV syringe),or a bag (e.g., an IV bag). A pharmaceutical composition in accordancewith the present disclosure may be prepared, packaged, and/or sold inbulk, as a single unit dose, and/or as a plurality of single unit doses.As used herein, a “unit dose” is discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient that would be administered to a subject and/or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. The examples below describe, in part, dosing ofexemplary IRTCA antibodies to rodents and monkeys. Standard methods areknown in the art of how to scale dosing in animal systems. See, forexample, J Basic Clin Pharm. March 2016-May 2016; 7(2): 27-31, which isincorporated herein by reference in its entirety. By way of example, thecomposition may comprise between 0.1% and 100% (w/w) active ingredient.

In some embodiments, a composition comprises or delivers an IRTCAantibody or antigen-binding fragment of the present disclosure at a doseof 0.01 mg/kg to 100 mg/kg. In some embodiments, a composition comprisesor delivers an IRTCA antibody or antigen-binding fragment at a dose inan amount within a range bounded by a lower limit and an upper limit,the upper limit being larger than the lower limit. In some embodiments,the lower limit may be about 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.075mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, 3mg/kg, 4 mg/kg, 5 mg/kg, 8 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg, 30mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, or 90 mg/kg. Insome embodiments, the upper limit may be about 0.025 mg/kg, 0.05 mg/kg,0.075 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 8 mg/kg, 10 mg/kg, 20 mg/kg, 25 mg/kg,30 mg/kg, 40 mg/kg, 50 mg/kg, 50 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, or100 mg/kg.

A pharmaceutical composition may additionally comprise apharmaceutically acceptable excipient, which, as used herein, includesany and all solvents, dispersion media, diluents, or other liquidvehicles, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21st Edition,A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006)discloses various excipients used in formulating pharmaceuticalcompositions and known techniques for the preparation thereof. Exceptinsofar as any conventional excipient medium is incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this disclosure.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humansand for veterinary use. In some embodiments, an excipient is approved bythe United States Food and Drug Administration. In some embodiments, anexcipient is pharmaceutical grade. In some embodiments, an excipientmeets the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical formulations.Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

In some embodiments, a provided pharmaceutical composition comprises oneor more pharmaceutically acceptable excipients (e.g., preservative,inert diluent, dispersing agent, surface active agent and/or emulsifier,buffering agent, etc.). In some embodiments, a pharmaceuticalcomposition comprises one or more preservatives. In some embodiments,pharmaceutical compositions comprise no preservative.

In some embodiments, a composition including an IRTCA antibody orantigen-binding fragment of the present disclosure is stably formulated.In some embodiments, a stable formulation of an IRTCA antibody orantigen-binding fragment of the present disclosure may comprise aphosphate buffer with saline or a chosen salt, as well as preservedsolutions and formulations containing a preservative as well asmulti-use preserved formulations suitable for pharmaceutical orveterinary use. Preserved formulations contain at least one knownpreservative or optionally selected from the group consisting of atleast one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzylalcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde,chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben(methyl, ethyl, propyl, butyl and the like), benzalkonium chloride,benzethonium chloride, sodium dehydroacetate and thimerosal, or mixturesthereof in an aqueous diluent. Any suitable concentration or mixture canbe used as known in the art, such as 0.001-5%, or any range or valuetherein, such as, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01,0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or valuetherein. Non-limiting examples include, no preservative, 0.1-2% m-cresol(e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5,0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005,0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%),0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005,0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75,0.9, 1.0%), and the like.

In some embodiments, a pharmaceutical composition is provided in a formthat can be refrigerated and/or frozen. In some embodiments, apharmaceutical composition is provided in a form that cannot berefrigerated and/or frozen. In some embodiments, reconstituted solutionsand/or liquid dosage forms may be stored for a certain period of timeafter reconstitution (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days,7 days, 10 days, 2 weeks, a month, two months, or longer). In someembodiments, storage of antibody compositions for longer than thespecified time results in antibody degradation.

Liquid dosage forms and/or reconstituted solutions may compriseparticulate matter and/or discoloration prior to administration. In someembodiments, a solution should not be used if discolored or cloudyand/or if particulate matter remains after filtration.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21st ed., Lippincott Williams &Wilkins, 2005.

Kits

The present disclosure further provides pharmaceutical packs and/or kitscomprising one or more containers filled with at least one IRTCAantibody or antibody fragment as described herein. Kits may be used inany applicable method, including, for example, therapeutic methods,diagnostic methods, cell proliferation and/or isolation methods, etc.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflects(a) approval by the agency of manufacture, use or sale for humanadministration, (b) directions for use, or both.

In some embodiments, a kit may include one or more reagents fordetection (e.g., detection of an IRTCA antibody or antibody fragment).In some embodiments, a kit may include an IRTCA antibody or antibodyfragment in a detectable form (e.g., covalently associated withdetectable moiety or entity).

In some embodiments, an IRTCA antibody or antibody fragment as providedherein may be included in a kit used for treatment of subjects. In someembodiments, an IRTCA antibody or antibody fragment as provided hereinmay be included in a kit used for conversion of T cells (e.g.,regulatory T cells converted to T_(H)1-like (IFN-γ-positive) cells).

Antibodies Having Reduced Tendency to Aggregate

An Isoelectric Point (pI) of a polypeptide such as an antibody or anantibody fragment indicates the pH at which the polypeptide carries nonet charge. One skilled in the art would appreciate that proteinsolubility is typically lowest when the pH of the solution is equal tothe isoelectric point (pI) of the protein. The pI of a protein may bedetermined by a variety of methods including but not limited to,isoelectric focusing and various computer algorithms (see, e.g.,Bjellqvist et al., 1993, Electrophoresis 14:1023).

In addition, the thermal unfolding (or melting) temperature (T_(m)) of apolypeptide such as an antibody can be a good indicator of the thermalstability of the polypeptide. A lower T_(m) typically indicatesincreased aggregation tendency of a polypeptide. T_(m) of a polypeptidesuch as an antibody can be measured using any standard method known inthe art, for example, by differential scanning calorimetry (see, e.g.,Vermeer et al., 2000, Biophys. J. 78:394-404; Vermeer et al., 2000,Biophys. J. 79: 2150-2154).

The present disclosure provides an IRTCA antibody or antibody fragmenthaving specific amino acid residue mutation(s) or modification(s) (e.g.,reduced oxidation, deamidation, glycation, or carbonylation) compared toa parent antibody or antibody fragment, wherein the mutation(s) ormodification(s) result in the antibody or antibody fragment havingreduced tendency to aggregate. For example, the anti-AITR antibodiesH1F1, H1F1M69, and H1F1M74 described herein have reduced tendency toaggregate as compared to the parent antibody IRTCA-A.

In some embodiments, the mutation(s) or modification(s) result in buriedhydrophobic surfaces or reduced β-sheet secondary structures in theantibody or antibody fragment when the antibody or antibody fragment isproperly folded.

In some embodiments, the mutation(s) or modification(s) result inchanges to the isoelectric point (pI) of the antibody or antibodyfragments.

In some embodiments, the mutation(s) or modification(s) results in anantibody that has an improved thermodynamic stability, for example,higher aggregation onset temperature (T_(agg)) or thermal unfoldingtemperature (T_(m)).

Various methods known in the art can be used to measure an antibody's orantibody fragment's tendency to aggregate. For example, an antibody orantibody fragment's tendency to aggregate measured using size exclusionchromatography or dynamic light scattering.

In some embodiments, the mutation(s) or modification(s) result in atleast a 0.1° C., 0.2° C., 0.3° C., 0.4° C., 0.5° C., 0.7° C., 1° C.,1.5° C., 2° C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C.,11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C.,or 20° C., increase in T_(m) or T_(agg) of the antibody or antibodyfragment, as compared to the T_(m) or T_(agg) of a parent antibody orantibody fragment.

In some embodiments, the mutation(s) or modification(s) result in atleast a 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% reduction inaggregate formation compared to a parent antibody or antibody fragment(e.g., as measured by size exclusion chromatography or dynamic lightscattering assays under the same or equivalent conditions).

In some embodiments, the mutation(s) or modification(s) result in achange of about −5, −4, −3, −2, −1, −0.7, −0.5, −0.4, −0.3, −0.2, −0.1,+0.1, +0.2, +0.3, +0.4, +0.5, +0.7, +1, +2, +3, +4, or +5 in pI of theantibody or antibody fragment as compared to a parent antibody orantibody fragment.

The contents of all cited references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments. However, thefollowing examples are merely provided to illustrate the presentinvention, but the scope of the present invention is not limited to thefollowing examples.

EXAMPLES

The present disclosure provides, at least in part, novel IRTCAantibodies and fragments thereof with high affinity toward hAITR,wherein binding of an exemplary IRTCA antibody to a T cell expressinghAITR on a T cell, can lead to immunity enhancement and/or anti-canceractivity. Generation and characterization of novel IRTCA antibodies andfragments thereof is described in further detail in the followingexamples.

Example 1: Cloning of Anti-AITR Antibodies, Including IRTCA-A

This example describes the production of exemplary IRTCA antibodies. Amonoclonal antibody (mAb), IRTCA-A, was used as a template, and eachheavy chain and light chain variable region gene and the human constantregion gene were amplified using polymerase chain reaction (PCR) beforetreatment with the Sfi-1 restriction enzyme, ligation, insertion into anexpression vector. The pelB leader sequence that mediates transfer to abacterial perioplasmic region was inserted upstream of a heavy chaingene and a signal peptide that induces secretion was inserted upstreamof a light chain gene for modification so that expression in E. colimight result in localization to the periplasmic region. The specificsequence of the IRTCA variable region was connected to a human kappachain for the light chain and connected with a human constant region forthe heavy chain to carry out cloning so that structural identity withnatural Fab might be maintained (see, FIG. 1A). The amino acid sequenceof the AITR (hGITR) derived epitope used in the present inventioninclude ‘HCGDPCCTTC’ (SEQ ID NO: 19), as determined in Example 16. Thiscorresponds to the 55^(th)-64^(th) amino acids of the extracellulardomain in AITR (hGITR).

Insertion of Amber Stop Codon in the Variable Regions of ParentalIRTCA-A

The technique of site-directed mutagenesis on one nucleotide was used togenerate amber stop codons in the immediately upstream of the CDR3region of the parental IRTCA-A variable region (that is, IRTCA-A:TGC->TGA). In IRTCA-A, the cysteine at position 89 in the parental formof the light chain and the cysteine at position 96 in the parental formof the heavy chain were mutated to a stop codon to build a CDR3randomized library. Selection of a parental sequence was prevented tolower the frequency of gene expression and to prevent contamination sothat screening might be efficiently achieved for designing (see, FIG.1B).

Example 2: Building of Anti-AITR Antibody Library and Fabrication ofAnti-AITR Antibody

Fabs comprising the parental base sequence of IRTCA-A, in which a stopcodon was inserted, were used as templates and randomized PCR wasperformed to afford random mutation in the CDR3 region (FIGS. 2A-2B).

While sequence changes in CDR3 do not result in significant changes inthe antigenic determinant of an antibody but increase variation, thepossibility of changing the epitope of the antibody to be selected alsoincreases in proportion with the number of amino acids with inducedmutation. Therefore, mutation rates of LCDR3-R and HCDR3-F primerprimers were modulated during the design of randomized PCR so thatoriginal epitopes recognized by IRTCA-A might not be modified and thatthe mutation rate of each amino acid sequence might be 70% or less. Forthe light chain, the splicing PCR technique was used to fix the heavychain sequence and fragment 1 (350 bp) amplified with omp primer andLCDR3-R and fragment 2 (˜1200 bp) amplified with LFR4-R and dp seq. wereused as templates to obtain fragment 3 of approximately 1500 bp whichhad random mutation in the light chain CDR3 region amplified with ompprimer and dp seq. (see, FIG. 2A). For the heavy chain, following theamplification conducted by the same mode (see, FIG. 2B) the resultingbase sequence was verified and then cloned into an expression vector toachieve expression on phage surface. That is, in order to minimizeinfluence on the antibody structure, under the condition that the lengthof the CDR3 (CDR-L3) region in a light variable chain and the length ofthe CDR3 (CDR-H3) region in a heavy chain were maintained. The frequencyof the clone which incurred mutation was measured and found to be ashigh as approximately 86%. In conclusion, a phage display library wasbuilt wherein diversity of light chain and heavy chain arrived 1×10⁸ orhigher (see, Table 1).

Table 1 shows variation of the phage library which expresses Fab ofIRTCA-A. That is, “Library diversity=transformant number×normal Fabcloning rate” may be used to calculate diversity.

TABLE 1 Transformant Intended Fab number cloning % rate DiversityIRTCA-A Light Chain 5.0 × 10⁸ 77.5 3.8 × 10⁸ IRTCA-A Heavy Chain 5.0 ×10⁸ 86.3 4.8 × 10⁸

On the other hand, in order to generate the anti-AITR antibody in abivalent form, Fab (VH (heavy chain variable region) and VL (light chainvariable region)) obtained from the phage display library was connectedwith the CH (heavy chain variable region) and CL (light chain variableregion) genes and then inserted in the protein expression vector foranimal cells. The vector into which the anti-AITR antibody gene wasinserted was transfected into animal cell strains including HEK293and/or CHO cells, and a protein A or protein G column was used to carryout purification from culture supernatant solution, which resulted inpreparation of the anti-human AITR monoclonal antibody in the bivalentform (data not shown).

Example 3: Selection of the Anti-AITR Antibody with Improved Affinity

The fabricated Fab (fragment antigen-binding) library was subject to aselection process with the fixed GST-AITR antigen and repeated fourtimes. For IRTCA-A, after repetitive panning was performed, 17 heavychain clones and 33 light chain clones with strong positive signals wereacquired. Since increase in affinity reduces K_(off) values, 47 Fabswere selected from a total of 50 clones and purified to measure K_(off)based on SPR (surface plasmon resonance) (see, FIG. 3 and FIG. 4). Forthe 5 (4 heavy chains, 1 light chain) Fabs with the decreased values,their K_(D) values were measured with SPR (surface plasmon resonance)(see, Table 2).

As indicated in Table 3, it was subsequently found that all of the finalIRTCA-A Fabs selected had increased affinity and that the rate ofincrease varied by 8-44 fold.

Table 2 lists K_(off) and K_(D) measurement values for five selectedspecies of IRTCA-A Fab against the AITR antigen.

TABLE 2 Clone K_(off) K_(D) Parental IRTCA-A 1.34 × 10⁻³-1.99 × 10⁻³1.14 × 10⁻⁷ IRTCA-A1 (Clone L1E9) 7.21 × 10⁻⁴ 1.39 × 10⁻⁸ IRTCA-A10(Clone H1A10) 5.64 × 10⁻⁴ 1.36 × 10⁻⁸ IRTCA-A12 (Clone H2A10) 3.91 ×10⁻⁴ 4.13 × 10⁻⁹ IRTCA-A14 (Clone H2F8) 3.45 × 10⁻⁴ 3.79 × 10⁻⁹IRTCA-A15 (Clone H1F1) 6.55 × 10⁻⁴ 2.57 × 10⁻⁹

Table 3 lists binding affinity measurements and fold ratio of 5 speciesof IRTCA-A Fab against the AITR antigen (K_(D)=K_(off)/K_(on), K_(D)ratio=mutant K_(D)/IRTCA-A K_(D)).

TABLE 3 Clone K_(on) (M⁻¹s⁻¹) K_(off) (s⁻¹) K_(A) (M⁻¹) K_(d) (M) Chi²K_(D) Ratio parental 1.17 × 10⁴ 1.34 × 10⁻³ 8.74 × 10⁶ 1.14 × 10⁻⁷ 0.6351.0 IRTCA-A H1A10 4.15 × 10⁴ 5.65 × 10⁻⁴ 7.34 × 10⁷ 1.36 × 10⁻⁸ 0.3998.38 H2A10 6.97 × 10⁴ 2.88 × 10⁻⁴ 2.42 × 10⁸ 4.13 × 10⁻⁹ 0.419 27.6 H1F11.30 × 10⁵ 3.34 × 10⁻⁴ 3.90 × 10⁸ 2.57 × 10⁻⁹ 0.934 44.4 H2F8 7.11 × 10⁴2.70 × 10⁻⁴ 2.64 × 10⁸ 3.79 × 10⁻⁹ 1.23 30.1 L1E9 2.96 × 10⁴ 4.12 × 10⁻⁴7.18 × 10⁷ 1.39 × 10⁻⁸ 0.641 8.2

Table 4 lists the SEQ ID NOs associated with the amino acid sequences ofthe heavy chain variable regions, light chain variable regions, heavychain CDRs and light chain CDRs for the clone antibodies in Table 2 toTable 3.

Table 5 lists the amino acid sequences of the heavy chain variableregions, light chain variable regions, heavy chain CDRs and light chainCDRs for the clone antibodies in Table 2 to Table 3.

TABLE 4 Heavy Light Chain Chain Variable Variable Heavy Chain LightChain Region Region HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 IRTCA SEQ ID SEQID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID Clone designation NO: NO:NO: NO: NO: NO: NO: NO: Parental 1 2 8 9 10 11 12 13 IRTCA-A L1E9IRTCA-A1 1 7 8 9 10 11 12 18 H1A10 IRTCA-A10 3 2 8 9 14 11 12 13 H2A10IRTCA-A12 4 2 8 9 15 11 12 13 H2F8 IRTCA-A14 6 2 8 9 17 11 12 13 H1F1IRTCA-A15 5 2 8 9 16 11 12 13

TABLE 5 Variable SEQ. Clone Region Amino Acid Sequence ID. ParentalHeavy QVQLVQSGTQVKMPGASVKVSCKASGYTFDDYGIGWVRQAPGQGLE 1 IRTCA-A ChainWMGWISPYTHRTNSSPKLQDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGTYYDFWSGYFDNGAFDIWGQGTLVTVSS LightQSVVTQPPSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQLPGTAPKLLIY 2 ChainDNYKRPSGIPDRFSGSKSGTSATLGITGLRTGDEADYFCGTWDSSLNAW VFGGGTKLTVL IRTCA-A1Heavy QVQLVQSGTQVKMPGASVKVSCKASGYTFDDYGIGWVRQAPGQGLE 1 (L1E9) ChainWMGWISPYTHRTNSSPKLQDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGTYYDFWSGYFDNGAFDIWGQGTLVTVS LightQSVVTQPPSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQLPGTAPKLLIY 7 ChainDNYKRPSGIPDRFSGSKSGTSATLGITGLRTGDEADYFCGSWESGSNAY KFGGGTKLTVL IRTCA-A10Heavy QVQLVQSGTQVKMPGASVKVSCKASGYTFDDYGIGWVRQAPGQGLE 3 (H1A10) ChainWMGWISPYTHRTNSSPKLQDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGTYYDFWSGYFDNAAFDSWGQGTLVTVSS LightQSVVTQPPSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQLPGTAPKLLIY 2 ChainDNYKRPSGIPDRFSGSKSGTSATLGITGLRTGDEADYFCGTWDSSLNAW VFGGGTKLTVL IRTCA-A12Heavy QVQLVQSGTQVKMPGASVKVSCKASGYTFDDYGIGWVRQAPGQGLE 4 (H2A10) ChainWMGWISPYTHRTNSSPKLQDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGTYYDFWSGYFDNATFDFWGQGTLVTVSS LightQSVVTQPPSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQLPGTAPKLLIY 2 ChainDNYKRPSGIPDRFSGSKSGTSATLGITGLRTGDEADYFCGTWDSSLNAW VFGGGTKLTVL IRTCA-A14Heavy QVQLVQSGTQVKMPGASVKVSCKASGYTFDDYGIGWVRQAPGQGLE 6 (H2F8) ChainWMGWISPYTHRTNSSPKLQDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGTYYDFWSGYFDTAAFDIWGQGTLVTVSS LightQSVVTQPPSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQLPGTAPKLLIY 2 ChainDNYKRPSGIPDRFSGSKSGTSATLGITGLRTGDEADYFCGTWDSSLNAW VFGGGTKLTVL IRTCA-A15Heavy QVQLVQSGTQVKMPGASVKVSCKASGYTFDDYGIGWVRQAPGQGLE 5 (H1F1) ChainWMGWISPYTHRTNSSPKLQDRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGTYYDFWSGYFDNNAFDIWGQGTLVTVSS LightQSVVTQPPSVSAAPGQKVTISCSGSTSNIGNNYVSWYQQLPGTAPKLLIY 2 ChainDNYKRPSGIPDRFSGSKSGTSATLGITGLRTGDEADYFCGTWDSSLNAW VFGGGTKLTVL

TABLE 6 CDR1 SEQ CDR2 SEQ CDR3 SEQ Variable Amino Acid ID Amino Acid IDAmino Acid ID Clone Region Sequence NO: Sequence NO: Sequence NO:Parental Heavy GYTFDDYG  8 ISPYTHRT  9 ARDGTYYDFWSGYFDN 10 IRTCA-A ChainGAFDI Light TSNIGNNY 11 DNY 12 GTWDSSLNAWV 13 Chain IRTCA-A1 HeavyGYTFDDYG  8 ISPYTHRT  9 ARDGTYYDFWSGYFDN 10 (L1E9) Chain GAFDI LightTSNIGNNY 11 DNY 12 GSWESGSNAYK 18 Chain IRTCA-A10 Heavy GYTFDDYG  8ISPYTHRT  9 ARDGTYYDFWSGYFDN 14 (H1A10) Chain AAFDS Light TSNIGNNY 11DNY 12 GTWDSSLNAWV 13 Chain IRTCA-A12 Heavy GYTFDDYG  8 ISPYTHRT  9ARDGTYYDFWSGYFDN 15 (H2A10) Chain ATFDF Light TSNIGNNY 11 DNY 12GTWDSSLNAWV 13 Chain IRTCA-A14 Heavy GYTFDDYG  8 ISPYTHRT  9ARDGTYYDFWSGYFDTA 17 (H2F8) Chain AFDI Light TSNIGNNY 11 DNY 12GTWDSSLNAWV 13 Chain IRTCA-A15 Heavy GYTFDDYG  8 ISPYTHRT  9ARDGTYYDFWSGYFDN 16 (H1F1) Chain NAFDI Light TSNIGNNY 11 DNY 12GTWDSSLNAWV 13 Chain

Example 4: Use of IFN-γ and TGF-β as Biomarkers

The T_(H)1 cell is evaluated to have the most efficient anti-cancercapacity among various T_(H) (helper T) cell types, and T_(reg) cell andTH2 cell are known to promote tumor formation by creatingimmunosuppressive environment around tumor. The T_(reg) cell is known tobe the suppressor T cell as well and responsible for resistance to autoantigen to suppress autoimmune diseases and excessive immune responses(Sakaguchi et al., Cell, 133:775-787, 2008). Accordingly, T_(reg) cellscan be useful in treating autoimmune diseases for this reason. On thecontrary, it weakens activity of the immune system against tumors cellsso that it decreases the anti-cancer capacity of a patient.Nevertheless, some subgroups in the T_(reg) cells whose expression ofFoxP3 is unstable may be converted into effector-memory phenotypes (Zhouet al., Nature immunology, 10:1000-1007, 2009). In addition, propertiesof the T_(reg) cell lacking VHL (Von Hippel-Lindau) are not fixed butmay undergo changes in a certain immune environment, for example, it canbe converted into the T_(eff) cell (effector T cell) which producesIFN-γ (Lee et al., Immunity, 42:1062-1074, 2015). Therefore,immunoregulation based on the T_(reg) cell represents the means ofeffective tumor suppression, and the technology of removing the T_(reg)cell or converting into the T_(eff) cell is the ultimate goal ofanti-cancer treatment with immune cell.

IFN-γ is the representative cytokine secreted from T-lymphocyte andnatural killer cell (NK cell) and exhibits proliferation and anti-viralactivity. It is also an important activator of macrophage andparticularly key cytokine which differentiates the T_(H)1 cell fromother cell types. Such T_(H)1 cell secretes IFN-γ to stimulate divisionby itself and has the capacity to differentiate the undifferentiatedCD4⁺ cell (T_(H)0) into the T_(H)1 cell. The differentiated T_(H)1 cellplays the key role in activating cytotoxic T cells, phagocytes and Bcells. On the other hand, TGF-β is known to be a very important factorfor immunoregulation of the Foxp3⁺ T_(reg) cell (Maria et al., Immunity,30:626-635, 2009), and in particular, promotes differentiation of theCD4⁺ T cell into the T_(reg) cell and T_(H)17 cell havingimmunosuppression capacities (Basso et al., Cell Res., 4:399-411, 2009).In the end, the efficiency of an anti-cancer drug can be determinedafter decrease in the T_(reg) cell secreting TGF-β and increase inT_(H)1 inducing IFN-γ must be relatively evaluated. For this reasonmeasurement of secretion of IFN-γ and TGF-β against certain stimulationmay be the optimal standard which can be utilized as the quantitativescale of change in the T cell function.

Thus in order to verify the potential of IFN-γ (interferon gamma) and/orTGF-β (transforming growth factor-beta) to serve as biomarkers anddetermine the activity of IRTCA-A against the T_(reg) cell,representative T_(reg) cell markers including CD4 and CD25 were used toisolate the CD4⁺CD25^(high)FOXP3⁺ T_(reg) cell from peripheral bloodmononuclear cells (PBMCs) and then to measure cytokine secretion by themonoclonal antibody (mAb).

Subsequently, as shown in FIG. 6A and FIG. 6B, IRTCA-A acted on AITR ofthe T_(reg) cell to increase secretion of IFN-γ and at the same timedecrease secretion of TGF-β. Additionally, IRTCA-A treatment inducedIFN-γ secretion in CD4⁺ T cell and polarized to T_(H)1 cells (FIG. 5).That is, IRTCA-A effectively stimulated AITR of the T cell in adose-dependent manner and promote conversion of T_(reg) into T_(H)1-likecells. This indicated that IFN-γ and TGF-β might be used as functionaltools with which to measure the effect of stimulating the AITR signalingsystem.

Example 5: Activation and Polarization of CD4⁺ T Cell Based onAffinity-Maturated IRTCA-A

This example describes the effects of affinity-matured IRTCA-A seriesantibodies on CD4⁺ T cells, including cytokine secretion.

First, in order to verify whether the mutation of Fabs changed theepitopes to which they could bind, a deletion mutant of AITR wasfabricated, and comparison with the parental form indicated that, asanticipated, the IRTCA-A series Fabs maintained their ability to bindthe AITR epitope (Table 7). In order to evaluate the functionality ofthe 5 species of affinity-maturated IRTCA-A Fab (IRTCA-A1, 10, 12, 14,15, of Table 7), which have high binding affinity with AITR compared toIRTCA-A, the polarization efficiency of the antibodies to change CD4⁺ Tcells into T_(H)1-type cells was measured.

Table 7 lists names of the Fab forms which were isolated from the Fabclones in the IRTCA-A series and then purified (*: Fab whoseepitope-binding ability was verified).

TABLE 7 Clone Name mAb Name IRTCA-A IRTCA-A L1E9 IRTCA-A1* H1A10IRTCA-A10* H2A10 IRTCA-A12* H2F8 IRTCA-A14* H1F1 IRTCA-A15*

As shown in Table 8 and FIG. 7A, when purified CD4⁺ T cells were treatedwith affinity-maturated mAb of IRTCA-A and the quantity of cytokinesecretion from culture supernatant was measured, affinity-maturated mAbspromote secretion of IFN-γ and also induce secretion of IFN-γ. Thesedata indicate that IRTCA has the function accelerating polarization ofCD4⁺ T cells into T_(H)1 cells secreting IFN-γ and that Fabs of the sameseries also interact with AITR on the surface of CD4⁺ T cells. Inparticular, administration of 0.5 μg IRTCA-A12, IRTCA-A14 and IRTCA-A15led cells to exhibit secretion of IFN-γ greater than the levels of IFN-γsecretion caused by IRTCA-A by approximately 200% or more (see, FIG. 7Aand Table 8).

Table 8 shows the results of measuring IFN-γ secretion in CD4⁺ T cellsafter administration of IRTCA-A series mAbs (K_(D) ratio=mutantK_(D)/IRTCA-A K_(D)).

TABLE 8 Clone K_(D) Ratio IFN-γ Secretion IRTCA-A 1.0 + IRTCA-A1 8.2 +IRTCA-A10 8.4 ++ IRTCA-A12 27.6 +++++ IRTCA-A14 30.1 +++++ IRTCA-A1544.4 +++++

Example 6: Effect of Anti-AITR Antibody on T_(reg) Cells (Regulatory TCells)

In order to determine influence of the mAbs which increased bindingaffinity in IRTCA-A on T_(reg) cells, CD4⁺CD25^(high)FOXP3⁺ T_(reg)cells were isolated and treated with IRTCA-A1, A10, A12, A14, and A15.

As shown in Table 9 and Table 10, after treatment with the IRTCA-Aantibodies, secretion of IFN-γ (a marker of T_(H)1 polarization)increased (FIG. 7B) whereas secretion of TGF-β (a representativesuppression cytokine of T_(reg) cells) decreased (FIG. 7C).

According to the above results, the mAbs of the IRTCA-A series areexpected to decrease the suppressive capacity of T_(reg) cells and toincrease activation of T_(eff) cells so that a micro-environmentcomprising anti-cancer effects is created. Additionally, as shown inTable 3, Table 9 and Table 10, increased affinity for AITR had asubstantial correlation with cytokine secretion, which contributes tothe anti-cancer role of the IRTCA-A antibodies. IRTCA-A12 and IRTCA-A15,which had smaller K_(D) values (see Table 3), that is, improved bindingaffinity with AITR compared with IRTCA-A, resulted in increasedsecretion of IFN-γ as well.

Furthermore, while the IRTCA-A series antibodies, which have differentCDR sequences are still similar to each other, bind to a common epitopeof the AITR antigen, it is also noteworthy that they exhibit effects ofdifferent tendency. In other words, it was anticipated that dependingupon binding affinity, IRTCA-A15 could achieve the same anti-cancereffect in the CD4⁺CD25^(high) T_(reg) cell at approximately 25% of thedose of IRTCA-A. This means not only that the dose of AITR mAb can beregulated to control the function of the T_(reg) cell, but also that adifferent mAb species that recognize the same epitope and but haveenhanced binding affinity can be used to control therapeutic effects bysending signals of different intensity.

Table 9 shows changes in secretion of IFN-γ from T_(reg) cells afteradministration of IRTCA-A series mAb.

TABLE 9 Clone K_(D) Ratio IFN-γ Secretion IRTCA-A 1.0 ++ IRTCA-A1 8.2 +IRTCA-A10 8.4 ++ IRTCA-A12 27.6 ++++ IRTCA-A14 30.1 + IRTCA-A15 44.4+++++

Table 10 shows changes in secretion of TGF-β from T_(reg) cells afteradministration of IRTCA-A series mAb.

TABLE 10 Clone K_(D) Ratio TGF-β Secretion IRTCA-A 1.0 +++++ IRTCA-A18.2 +++ IRTCA-A10 8.4 +++ IRTCA-A12 27.6 + IRTCA-A14 30.1 + IRTCA-A1544.4 +

Example 7: Engraftment of Human Peripheral Blood Mononuclear Cells inNOD-SCID Mouse and Anti-Tumor Activity of Anti-AITR Antibody

This example describes the anti-tumor effects of anti-AITR antibodies onNOD-SCID mice after administration of human peripheral blood mononuclearcells (PBMCs) and human colorectal cancer cells.

First, after peripheral venous blood collected from healthy donors withthe HLA-A24 type was treated with heparin, Ficoll-paque (GE Healthcare,Piscataway, N.J.) density gradient centrifugal separation was conductedto obtain peripheral blood mononuclear cells (PBMCs). Next, within 3hours used for recovery, 1×10⁷ human PBMCs included in RPMI was injectedinto each NOD-SCID mouse peritoneally. The 21 grafted mice above wereanalyzed in 5 weeks following injection of human PBMCs (HLA-A24 type).

The 7-week-old NOD-SCID mice (Jackson Laboratory, Barharbor, Me.) werebred in an animal room that was a SPF (specific pathogen free)environment. All animal experiments were performed according to theExperimental Animal Ethics Guidelines.

In order to evaluate the anti-cancer effects of the anti-AITR monoclonalantibodies (IRTCA), human colorectal cancer cells of HT29 (HLA-A24 type,1×10⁷ cells/mouse) were subcutaneously injected on the dorsal side ofthe Hu-PBL-SCID mouse, and when the tumor size reached the diameter of˜2 cm, the anti-AITR monoclonal antibody (IRTCA) was administeredintraperitoneally at doses of 0.3 mg, 0.6 mg, 1.0 mg, 3.0 mg, 5.0 mg and10.0 mg per 1 kg body weight every 5 days, for a total of 3administrations. Human IgG was used as the control group. The mousetumor volume (in mm³) was measured according to the group and date.

As shown in FIG. 8, the tumor size in mice treated with the anti-AITRmonoclonal antibodies (IRTCA) decreased in a manner that correlated withantibody concentration. In particular, the IRTCA concentration of 3.0mg/kg revealed such effect that the tumors were completely eradiated byDay 33.

Additionally, shown in FIG. 9, when tumor-challenged mice injected withHT29 cells were treated with control hIgG, H1F1, MK4166 (Merck'santi-GITR antibody) or Pambrolizumab (Keytruda) via i.v. injection atone week intervals at a dosage of 3.0 mg/kg, only H1F1 resulted in adecrease in tumor size over several weeks.

The exemplary anti-AITR monoclonal antibody H1F1 was also administeredto 7-weeks-old, female, NOD-SCID mice humanized with human PBMCs (asdescribed above), in order to test its anti-tumor effect against fourcancers: triple negative breast cancer (MDA-MB231), colon cancer (HT-29)and melanoma (MDA-MB435, SK-MEL2). Mice were injected with 1×10⁷ tumorcells per mouse and once the size of the tumors reached 1000-1500 mm³ involume, the tumor-challenged mice were injected intraperitoneally (i.p.)with H1F1 or hIgG once every three days for three times. The width,length, and height of the tumors were measured with calipers at the timepoints indicated in FIGS. 10A-10D. The arrows indicate the time pointswhen H1F1 or control were injected.

Example 8: Analysis of Tissue Cross Reactivity, Toxicology & Mechanismof Action of Anti-AITR Antibody in Cynomolgus Monkey

This example describes the effects of treatment with an exemplaryanti-AITR antibody on cynomolgus monkeys. Seven Cynomolgus monkeys(Macaca fascicularis) approximately 25-35 years of age and approximately2-3 kg in weight were used for this study. The control group contained 1female monkey who received a dose volume of 39 mL/kg. The low dose groupcontained 1 male monkey and 1 female monkey who each received an H1F1dosage of 22.5 mg/kg at a concentration of 2.3 mg/kg and a dose volumeof 9.75 mL/kg. The middle dose group contained 1 male monkey and 1female monkey who each received an H1F1 dosage of 45 mg/kg at aconcentration of 2.3 mg/kg and a dose volume of 19.5 mL/kg. The highdose group contained 1 male monkey and 1 female monkey who each receivedan H1F1 dosage of 90 mg/kg at a concentration of 2.3 mg/kg and a dosevolume of 39 mL/kg.

When toxicology tests were run on the monkeys, no abnormalities wereobserved in clinical symptoms, body/organ weight, blood tests, urinetests, histopathological examination or electrocadiography. Theno-observed-adverse-effect-level (NOAEL) for H1F1 was found to be over90 mg/kg in macaques.

In vitro, the effect of H1F1 on cytokine secretion was tested onCD4⁺CD25^(high)Foxp3⁺ T cells. As shown in FIG. 11, treatment with H1F1lead to polarization of CD4⁺CD25^(high)Foxp3⁺ T cells to T_(H)1 cells,evidenced by an increase in the secretion of IFN-γ and a decrease in theTGF-β after treatment with H1F1.

An H1F1-mediated conversion of regulatory T cells to T_(H)1 cells wasalso observed in macaques in vivo. As shown in FIGS. 12A-12D, all threedoses of H1F1 lead to an increase in IFN-γ positive CD4⁺T cells over thecourse of 3 weeks. Additionally, H1F1 cytokine induction wasdifferential, as shown in FIGS. 13A-13D, T_(H)1-type of cytokines (IL-2and IFN-γ) were increased after treatment with H1F1, whereas the levelof TH2-type cytokines (IL-4 and IL-5) were not altered. Treatment withH1F1 also affected the balance between T_(reg) and T_(H)1 cells inmacaques in vivo. As shown in FIGS. 14A-14D, in splenocytes ofH1F1-administered macaques, the population of regulatory T cells and theassociated cytokine TGF-β were decreased, while the population ofT_(H)1-type cells increased.

Additionally, tissue cross-reactivity studies were performed on tissuesfrom 15 different rat organs, 16 different dog organs, 16 differentmonkey organs, 13 different human organs (138 different tissues) andtissues from cancer patients with EBV-associated gastric cancer and bonemarrow from acute myeloid leukemia. H1F1-specific staining was detectedin the immune system but not in other normal human tissues. FIG. 15shows that H1F1-specific staining was observed in EBV-positive gastriccancer and bone marrow from an AML patient. Cross-reactivity wasdetected in macaque and dog lymphocytes, but no cross-reactivity wasobserved in rat tissues.

Example 9: Creation of Improved Anti-AITR Antibodies

This example describes the production of optimized anti-AITR antibodies.

Anti-AITR Antibody Production

Expi293 cells were put in a 125 mL Erlenmeyer flask and cultured inExpi293 expression medium in an 8% CO₂ incubator under spin (125 rpm) at37° C. Cells were cultured at 2-2.5×10⁶ cell/ml in flask. On day −1,cells (2×10⁶ cell/ml per flask) were prepared in 30 ml of Expi293expression medium in a 125 mL Erlenmeyer flask. On day 0, Opti-MEMmedium was pre-warmed in a 37° C. incubator for about 30 minutes. Tubes1 and 2 were prepared, and 1.5 mL of pre-warmed Opti-MEM medium wasadded to each tube. 30 μg of DNA (HC: 15 μg, LC: 15 μg) were added toTube 1 and mixed well. 80 μL of Expifectamine 293 reagent was added toTube 2 and mixed well. After reaction for 5 minutes at room temperature,the contents of Tube 2 were added to Tube 1 and mixed well. The mixturewas allowed to react for 20 minutes at room temperature. Once themixture in the tube was confirmed to have turned cloudy, 3 mL of theDNA-Complex was taken from the flask and added dropwise. After adding,it was cultured in an incubator for 18-20 minutes while being spun, andenhancer 1 and enhancer 2 provided in the Expifectamine 293 Transfectionkit were added. It was cultured while being spun for about 7 days. Table11 below summarizes the heavy (HC) variable domains and light chain (LC)variable domains used to create the mutant anti-AITR antibodies. Table12 below includes the heavy chain and light chain variable domain andCDR SEQ ID NOs corresponding to the mutant anti-AITR antibodies.

TABLE 11 H1F1 Mutant antibody Expi293 cell transfection table Ab HC LCH1F1.M1 H1F1 WT H1F1.L1 H1F1.M2 H1F1 WT H1F1.L2 H1F1.M3 H1F1 WT H1F1.L3H1F1.M4 H1F1.H1 H1F1.WT H1F1.M5 H1F1.H1 H1F1.L1 H1F1.M6 H1F1.H1 H1F1.L2H1F1.M7 H1F1.H1 H1F1.L3 H1F1.M8 H1F1.H2 H1F1.WT H1F1.M9 H1F1.H2 H1F1.L1H1F1.M10 H1F1.H2 H1F1.L2 H1F1.M11 H1F1.H2 H1F1.L3 H1F1.M12 H1F1.H3H1F1.WT H1F1.M13 H1F1.H3 H1F1.L1 H1F1.M14 H1F1.H3 H1F1.L2 H1F1.M15H1F1.H3 H1F1.L3 H1F1.M16 H1F1.H4 H1F1.WT H1F1.M17 H1F1.H4 H1F1.L1H1F1.M18 H1F1.H4 H1F1.L2 H1F1.M19 H1F1.H4 H1F1.L3 H1F1.M20 H1F1.H5H1F1.WT H1F1.M21 H1F1.H5 H1F1.L1 H1F1.M22 H1F1.H5 H1F1.L2 H1F1.M23H1F1.H5 H1F1.L3 H1F1.M24 H1F1.H2 LC-A1 H1F1.M25 H1F1.H2 LC-A2 H1F1.M26H1F1.H2 LC-A3 H1F1.M27 H1F1.H2 LC-A4 H1F1.M28 H1F1.H2 LC-A5 H1F1.M29H1F1.H2 LC-A6 H1F1.M30 H1F1.H2 LC-A7 H1F1.M31 H1F1.WT.HC.SHH1F1.WT.LC.SH H1F1.M32 JLHC1.A.HC.SH JLHC1.A.LC.SH H1F1.M33H1F1.OM.A.HC.SH H1F1.OM.A.LC.SH H1F1.M34 H1F1.H2 H1F1.L4 H1F1.M35H1F1.H6 H1F1.WT.LC.SH H1F1.M36 H1F1.H6 H1F1.L2 H1F1.M37 H1F1.H6 H1F1.L4H1F1.M38 H1F1.H7 H1F1.L5 H1F1.M39 H1F1.H7 H1F1.L6 H1F1.M40 H1F1.H8H1F1.L5 H1F1.M41 H1F1.H8 H1F1.L6 H1F1.M42 H1F1.H2.J1 H1F1.WT(A27VL)H1F1.M43 H1F1.H2.J1 H1F1.L2 H1F1.M44 H1F1.H2.J1 H1F1.L3 H1F1.M45H1F1.H2.J1 H1F1.L4 H1F1.M46 H1F1.H2.J1 H1F1.L5 H1F1.M47 H1F1.H2.J1H1F1.L6 H1F1.M48 H1F1.H2 H1F1.L3.J3 H1F1.M49 H1F1.H2.J1 H1F1.L3.J3H1F1.M50 H1F1.H6 H1F1.L3.J3 H1F1.M51 H1F1.H7 H1F1.L3.J3 H1F1.M52 H1F1.H8H1F1.L3.J3 H1F1.M53 H1F1.H2.J2 H1F1.WT(A27VL) H1F1.M54 H1F1.H2.J2H1F1.L2 H1F1.M55 H1F1.H2.J2 H1F1.L4 H1F1.M56 H1F1.H2.J2 H1F1.L5 H1F1.M57H1F1.H2.J2 H1F1.L6 H1F1.M58 H1F1.H2 H1F1.L3.J1 H1F1.M59 H1F1.H6H1F1.L3.J1 H1F1.M60 H1F1.H7 H1F1.L3.J1 H1F1.M61 H1F1.H8 H1F1.L3.J1H1F1.M62 H1F1.H2.J1 H1F1.L3.J1 H1F1.M63 H1F1.H2.J2 H1F1.L3.J1 H1F1.M64H1F1.H2.J1 H1F1.WT.LC.SH H1F1.M65 H1F1.H2.J2 H1F1.WT.LC.SH H1F1.M66H1F1.WT.HC.SH H1F1.L3.J1 H1F1.M67 H1F1.WT.HC.SH H1F1.L3.J3 H1F1.M68H1F1.H2.J2 H1F1.L3.J3 H1F1.M69 H1F1.H2.J1 H1F1.L3.J1 H1F1.M70 H1F1.H2.J2H1F1.L3.J1 H1F1.M71 H1F1.H8 H1F1.L3.J1 H1F1.M72 H1F1.H2.J1 H1F1.L3.J2H1F1.M73 H1F1.H2.J2 H1F1.L3.J2 H1F1.M74 H1F1.H8 H1F1.L3.J2

TABLE 12 Heavy Light Chain Chain Variable Variable Heavy Chain LightChain Region Region HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 mAb name SEQ IDSEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID (Clone) NO: NO: NO: NO:NO: NO: NO: NO: H1F1.M69 21 22 24 25 16 11 12 13 (H1F1.H2.J1/H1F1.L3.J1) H1F1.M74 20 23 8 25 16 11 12 18 (H1F1.H8/ H1F1.L3.J2)

H1F1 Mutant Antibody Sequences H1F1.H8 (SEQ ID NO: 20)QVQLVQSGAQVKMPGESLKVSCKASGYTFDDYGMGWVRQAPGQCLEWMGWISPYTGRTNSSDKFQGRVTMTRDTSTSTAYMELRSLRSEDTAVYYCARDGTYYDFWSGYFDNNAFDIWGQGTLVTVSS H1F1.H2.J1 (SEQ ID NO: 21)QVQLVQSGAQVKMPGESLKVSCKASGYTFTDYGMGWVRQAPGQGLEWMGWISPYTGRTNSSDKFQGRVTMTRDTSTSTAYMELRSLRSEDTAVYYCARDGTYYDFWSGYFDNNAFDIWGQGTLVTVSS H1F1.L3.J1 (SEQ ID NO: 22)QSVVTQPPSVSGAPGQRVTISCSGSTSNIGNNYVSWYQQLPGTAPKLLIYDNYKRPSGVPDRFSGSKSGTSASLAITGLQTEDEADYYCGTWDSSLNAWV FGGGTKLTVL H1F1.L3.J2(SEQ ID NO: 23) QSVVTQPPSVSGAPGQRVTISCSGSTSNIGNNYVSWYQQLPGTAPKLLIYDNYKRPSGVPDRFSGSKSGTSASLAITGLQSEDEADYYCGSWESGSNAYK FGGGTKLTVLCDR1 of H1F1.H2.J1 (SEQ ID NO: 24) GYTFTDYGCDR2 of H1F1.H2.J1 and H1F1.H8 (SEQ ID NO: 25) ISPYTGRT  hIgG1_3mu(SEQ ID NO: 26) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Anti-AITR Antibody Purification

Chromatography Column Preparation

The column (20 ml-volume) was filled with 20% ethanol and washed once.The column was filled with binding buffer and washed once. Any remainingbuffer was removed completely from the column using a pipette. 3-5 ml of20% ethanol was added, and 1 ml of Mab Select SuRe LX resin was addedslowly, with the amount of eluting ethanol was checked during theprocedure. The procedure continued until all of 20% ethanol was drainedand Select SuRe LX resin reached 2 ml.

Preparation of Antibody Mixture

The antibody mixture obtained from transfected Expi293 cells werecentrifuged (10,000 rpm, 10 min), and cells and impurities were pelleted(10,000 rpm, 10 min). The supernatant was filtered using a 0.22 μmsyringe filter.

Antibody Purification (for Open-Column)

20% ethanol filled in the column packaged with Mab Select SuRe LX wasdrained completely, and the column was washed with 18 ml of Bindingbuffer once. The entire antibody mixture was loaded. The column waswashed with 18 ml Binding buffer once. Once the buffer was drainedcompletely, a 1.5 ml tube with 1000 of the neutralization buffer wasplaced under the column, and antibodies attached to the column werepurified using 1 ml of Elution buffer each time.

Antibody Purification (for FPLC)

The Mab select SURE (1 ml) column was connected to the FPLC andstabilized with buffer A (1×PBS). After stabilizing until no other peakswere detected, antibody binding was performed by applying the sample tothe column at the flow rate of 1 ml/min. Apply Buffer A to the columnuntil the UV graph increased from approximately 1500 mAU to 2000 mAU anddecreased to below 10 mAU. After this, antibodies were eluted byapplying Buffer B (0.1M glycine pH 3.0), and the elution peak wasconfirmed by the UV graph. The fraction of the applicable peak wasobtained.

Antibody Dialysis

After antibody purification, 5 μl of Bradford solution was added to 250μl of the eluted sample for a color reaction, and the antibody productwas concentrated to a volume between 1-1.5 ml using 4 units of AmiconUltra filters. The concentrated antibody product was added to a conicaltube with 43 ml of 1×DPBS in a Dialysis device and dialyzed using ashaker (120 rpm, 2 hr). This step was repeated 2 times by replacing withnew 1×DPBS. The antibody product was moved from the dialysis device to aSpin-X centrifuge tube filter unit, centrifuged (10000 rpm, 2 min), putinto a 2 ml storage glass bottle, and stored at −20° C. and −80° C.until use.

Example 10: Characterization of Improved Anti-AITR AntibodiesDetermining AITR Binding Affinity of Improved Anti-AITR Antibodies

A cell line overexpressing AITR was cultured in complete medium(H-DMEM+10% FBS+1× Penicillin-Streptomycin) at 37° C. in a 5% CO₂incubator. Subcultures were made every 3 days while monitoring themorphology, number, and growth rate of the cell line.

Once cells were prepared using a 1× Trypsin/EDTA solution of the cellline overexpressing AITR, cells were aliquoted at 5×10⁵ per FACS tube.After adding 10 μg, 5 μg, 2.5 μg, or 1.25 μg of the H1F1 mutantantibody, the reaction was performed for 30 min at 4° C. After adding 3ml of FACS buffer (1×DPBS+1% FBS), centrifugation, and washing, asecondary antibody (Goat Anti-Human DyLight® 488) was added andincubated at 2-8° C. for 30 minutes. After centrifuging and washing with3 mL of FACS buffer, FACS analysis was performed after adding FACSstorage buffer (1×DPBS+2.5% FBS). For controls, cells withoutantibodies, cells with secondary antibody only, and cells with AITR-PEantibody were used.

Example 11: Analysis of Binding Ability Between AITR Antigen andAnti-AITR Antibodies H1F1M69 and H1F1M74

This example assesses the ability of mutant anti-AITR antibodies to bindto an AITR antigen recognized by the parent antibody, H1F1. Surfaceplasmon resonance was used in order to determine the ability of mutantanti-AITR antibodies to bind to AITR.

A Biacore T200 with a CMS sensor chip was run using HBS-EP runningbuffer at pH 7.4 and 3M magnesium chloride regeneration buffer.Anti-human IgG(Fc) antibodies were immobilized to the CMS chip via aminecoupling and 6 minute injections at a flow rate of 5 μL/minute wereperformed with an immobilization buffer of 10 mM sodium acetate at pH5.0. The capture antibodies tested were parent antibody H1F1 and mutantantibodies M69 and M74. Glutathione S-transferase (GST)-tagged AITRanalyte was injected at concentrations of 3.12, 6.25, 12.5, 25, 50 and100 nM, with an association time of 150 seconds and a dissociation timeof 240 seconds. Regeneration was achieved with 3M magnesium chloride for30 seconds and 1:1 74binding was evaluated.

As shown in Table 13 below, H1F1 mutants M69 and M74 bound the GST-AITRanalyte with an affinity comparable to that of the parent antibody H1F1.

TABLE 13 Antibody K_(D) (M) H1F1M69 6.574 × 10⁻¹⁰ H1F1M74 9.655 × 10⁻¹⁰H1F1 7.892 × 10⁻¹⁰

The anti-AITR antibodies M69 and M74 were further analyzed for bindingto AITR using a HEK293 cell line that overexpressed human AITR (AITR-5).As shown in FIG. 16A, the negative control mAb EU101 did not bind toAITR-5 (3.6%), while 89.8% of AITR-5 cells were stained with H1F1 in 10μg/mL of concentration. Additionally, as shown in FIG. 16B, 89.1% ofAITR-5 cells were stained with M69 and 73.8% of the cells were stainedwith M74. These data indicate that H1F1, M69 and M74 all bind to thehuman AITR molecule that was expressed on the cell surface in a dosedependent manner.

Example 12: Effect of Anti-AITR Antibodies H1F1M69 and H1F1M74 onnT_(reg) Cells (Regulatory T Cells)

In this example, mutant anti-AITR antibodies were tested for theirability to convert nT_(reg) cells to T_(H)1 cells.

Peripheral blood mononuclear cells (PBMCs) were collected viaFicoll-Paque density gradient centrifugation and then FACS was used toisolate CD4⁺CD25⁺⁺CD127⁻ (nT_(reg)) cells. The nT_(reg) cells were thentreated for 5 days with 100 U/mL IL-2 every 2-3 days and with monoclonalantibodies H1F1M69, H1F1M74, TRX518, and MK4166 at concentrations of 2.5and 10 μg/mL, with 10 μg/mL of hIgG used as a negative control. Afterthe 5 days of treatment, the cells were tested for intracellularstaining of IFN-γ and by ELISA for IFN-γ and TGF-β secretion.

As shown in FIGS. 17A-17C, the nT_(reg) cells were converted toT_(H)1-like cells upon stimulation with H1F1M69 and H1F1M74. 71% ofT_(reg) were converted to IFN-γ-positive cells by M69 and 57% of T_(reg)were converted to IFN-γ-positive cells by M74. The numbers in each panelindicate the percentage of positive cells.

For testing secretion of IFN-γ and TGF-β from T cells after stimulationwith IL-2 and anti-AITR antibodies, culture supernatants were used tomeasure IFN-γ and TGF-β. The graph in FIG. 18 summarizes the resultsobtained by ELISA assay. H1F1M69 and H1F1M74 stimulated secretion of theT_(H)1 cytokine, IFN-γ (gray bars) and, conversely, lead to decreasedsecretion of the T_(reg) cytokine, TGF-β (black bars). 5×10⁴ nT_(reg)cells per well were used for this assay.

Example 13: Effect of Anti-AITR Antibodies H1F1M69 and H1F1M74 oniT_(reg) Cells (Inducible Regulatory T Cells)

In this example, mutant anti-AITR antibodies were tested for theirability to convert inducible T_(reg) (iT_(reg)) cells to T_(H)1 cells.

Peripheral blood mononuclear cells (PBMCs) were collected viaFicoll-Paque density gradient centrifugation and then magnetic-activatedcell sorting (MACS) with CD4 microbeads (Miltenyi) was used to isolateCD4⁺ T cells according to the manufacturer's instructions. The CD4⁺ Tcells were then stimulated for 6 days with anti-CD3/anti-CD28 beads (25μL, Invitrogen), 100 U/mL IL-2 (Novartis) and 5 ng/mL TGF-β1. After the6 days of stimulation, the cells were then stained for CD4-BB515 andFoxp3-APC. The CD3/CD28 beads were then removed via MACS. The CD4⁺ Tcells were then treated for 5 days with 100 U/mL IL-2 every 2-3 days andwith monoclonal antibodies H1F1M69, H1F1M74, TRX518, and MK4166 atconcentrations of 2.5, 5, and 10 μg/mL, with 10 μg/mL of hIgG used as anegative control. After the 5 days of treatment, the cells were testedfor intracellular staining of IFN-γ and by ELISA for IFN-γ and TGF-βsecretion.

The transcription factor forkhead box protein P3 (Foxp3) is a specificmarker of regulatory T cells and plays a pivotal role in the suppressiveactivity of nT_(reg) and iT_(reg) cells. To evaluate effect of AITRsignaling in T_(reg), regulatory T cells were induced in vitro and Foxp3expression was measured. As shown in FIG. 19, iT_(reg) cells weregenerated from the CD4⁺ T cells after stimulation with anti-CD3, CD28beads, IL-2 and TGF-β for 6 days. 83.7% of iT_(reg) were confirmed byFoxp3-positive staining (right panel). These data indicate that the CD4⁺T cell population was differentiated into iTreg by the conditionsdescribed above.

Although AITR expression on CD4⁺ T cells is dependent on activation,naïve or inducible regulatory T cell express AITR constitutively. Priorto measuring Foxp3 expression, the binding efficiency of H1F1 for AITRon iT_(reg) cells was determined. As shown in FIGS. 20A-20G, surfaceAITR was detected by mutant H1F1 antibodies (M69—FIG. 20D and M74—FIG.20E). Furthermore, H1F1M69 and H1F1M74 recognized approximately 39% ofthe cells as positive for human AITR, which was more frequently thancompetitor's anti-AITR antibodies (19% of cells in average—FIGS. 20F and20G).

Additionally, as shown in FIGS. 21A-20E, the iT_(reg) cells wereconverted to T_(H)1-like cells upon stimulation with H1F1M69 andH1F1M74. 78% of iT_(reg) were converted to IFN-γ-positive cells by M69and 57% of iT_(reg) were converted to IFN-γ-positive cells by M74. Thenumbers in each panel indicate the percentage of positive cells.

For testing secretion of IFN-γ and TGF-β from T cells after stimulationwith IL-2 and anti-AITR antibodies, culture supernatants were used tomeasure IFN-γ and TGF-β. The graph in FIG. 22 summarizes the resultsobtained by ELISA assay. H1F1M69 and H1F1M74 stimulated secretion of theT_(H)1 cytokine, IFN-γ (gray bars) and, conversely, lead to decreasedsecretion of the T_(reg) cytokine, TGF-β (black bars). 1×10⁵ iT_(reg)cells per well were used for this assay.

Example 14: Effect of Immobilized Anti-AITR Antibodies H1F1M69 andH1F1M74 on iT_(reg) Cells (Inducible Regulatory T Cells)

In this example, immobilized mutant anti-AITR antibodies were tested fortheir ability to convert inducible T_(reg) (iT_(reg)) cells to T_(H)1cells.

Peripheral blood mononuclear cells (PBMCs) were collected viaFicoll-Paque density gradient centrifugation and then magnetic-activatedcell sorting (MACS) with CD4 microbeads (Miltenyi) was used to isolateCD4⁺ T cells according to the manufacturer's instructions. The CD4⁺ Tcells were then stimulated for 6 days with anti-CD3/anti-CD28 beads (25μL, Invitrogen), 100 U/mL IL-2 (Novartis) and 5 ng/mL TGF-β1. After the6 days of stimulation, the cells were then stained for CD4-BB515 andFoxp3-APC. The CD3/CD28 beads were then removed via MACS. The CD4⁺ Tcells were then treated over 5 days with 100 U/mL IL-2 every 2-3 daysand with immobilized monoclonal antibodies M69 and M74 at concentrationsof 2.5, 5, and 10 μg/mL for approximately 1 day. After the 5 days oftreatment, the cells were tested for intracellular staining of IFN-γ andby ELISA for IFN-γ and TGF-β secretion.

Since down-regulation of Foxp3 reduces suppressive activity of iT_(reg),iT_(reg) cells were prepared and the effect of H1F1M69 and H1F1M74 onexpression of Foxp3 was evaluated. As described above, to creatediT_(reg) cells, CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28beads and polarized to iT_(reg) in vitro. As shown in FIGS. 23B and 23C,immobilized H1F1M69 and H1F1M74 inhibited expression of Foxp3 iniT_(reg). In addition, although TRX518 or MK4166 failed to reduce Foxp3expression (FIGS. 23D-23E), H1F1M69 and H1F1M74 decreased Foxp3expression even at their lowest concentrations (2.5 μg/mL). Thus,H1F1M69 and H1F1M74 induced loss of Foxp3, which is an essentialtranscription factor for maintaining immune-suppressive activity ofregulatory T cell.

As shown in FIGS. 24A-24B, the iT_(reg) cells were converted toT_(H)1-like cells after being added to wells coated with IL-2 and theanti-AITR antibodies H1F1M69 and H1F1M74. 90% of iT_(reg) were convertedto IFN-γ-positive cells by M69 and 68% of iT_(reg) were converted toIFN-γ-positive cells by M74 in a dose-dependent manner. The numbers ineach panel indicate the percentage of positive cells.

Example 15: Effect of Anti-AITR Antibodies M69 and M74 on T_(eff) Cells(Effector T Cells)

In this example, mutant anti-AITR antibodies were tested for theirability to convert T_(eff) cells to T_(H)1 cells.

Whole blood was collected from healthy donors and PBMCs were separatedout in the buffy coat by Ficoll-Paque density gradient centrifugation.PBMCs were collected from the interface between the plasma layer and theFicoll-RBC layer and washed with RPMI1640 media. PBMCs were resuspendedwith pre-sorting buffer and stained with anti-human CD4, anti-CD25 andanti-CD127. CD4⁺CD25⁻CD127⁺ (T_(eff)) cells were sorted by FACS.

As shown in FIGS. 25A-25C, the anti-AITR antibodies H1F1M69 and H1F1M74polarized the T_(eff) cells to T_(H)1 cells. 81.1% of T_(eff) cells werepolarized to IFN-γ-positive cells by H1F1M69 (10 μg/mL) and 75.3% ofT_(eff) cells were polarized to IFN-γ-positive cells by H1F1M74 (10μg/mL). The numbers in each panel indicate the percentage of positivecells.

Additionally, the polarized T_(eff) cells were tested for IFN-γsecretion. The graph in FIG. 26 summarizes the results obtained by ELISAassay. H1F1M69 and H1F1M74 stimulated secretion of the T_(H)1 cytokine,IFN-γ, indicating that the T_(eff) cells were polarized to T_(H)1 cells.

Example 16: Epitope Mapping of Anti-AITR Antibodies IRTCA-A (A27), H1F1,M69 and M74

In this example, epitope mapping of the anti-AITR antibodies IRTCA-A,H1F1, H1F1M69 and H1F1M74 were performed by assessing their ability tobind to GST fusion protein constructs containing overlapping portions ofthe extracellular domain of AITR (R1-R12).

Plasmid constructs encoding the GST fusion protein constructs R1-R12 (asshown in FIG. 27A) were prepared by inserting constructs encodingportions of the extracellular domain of AITR (obtained from Bioneer,South Korea) into pBT7-based vector. Fusion proteins were expressed inE. coli with IPTG (0.50 mM) and purified from bacterial lysates byaffinity chromatography using Ni-Sepharose High Performance (GEhealthcare, Uppsala, Sweden).

The presence of the GST-fusion proteins was confirmed by Western blotanalysis using 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) andcoomassie blue staining and anti-GST (1:10000; Abcam, Abcam, Cambridge,Mass., USA) immunoblotting.

Binding of the anti-AITR antibodies IRTCA-A (A27), H1F1, H1F1M69 andH1F1M74 to the GST-fusion proteins R1-R12 was assessed using Westernblot analysis, in which the GST-fusion proteins were resolved by 12%SDS-PAGE and subjected to immunoblotting using the antibodies IRTCA-A,H1F1, H1F1M69 and H1F1M74. Anti-Human IgG-HRP (1:5000; JacksonLaboratories, Bar Harbor, Me.) was used as the secondary antibody.Detection of chemiluminescence was obtained by using SuperSignal WestPico Chemiluminescent Substrate (Pierce, Rockford, Ill.).

Binding of the anti-AITR antibodies IRTCA-A (A27), H1F1, H1F1M69 andH1F1M74 to the GST-fusion proteins R1-R12 was also confirmed using ELISAanalysis, in which the proteins were coated on 96 well immune plate andtreated using the antibodies IRTCA-A, H1F1, M69 and M74. Anti-HumanIgG-HRP antibody (1:5000; Jackson Laboratories, Bar Harbor, Me.) oranti-GST antibody (1:10000; Abcam, Abcam, Cambridge, Mass., USA) wasused as the secondary antibody. For substrate solution, BD OptEIA TMBSubstrate (BD Biosciences, San Diego, Calif., USA) was used and thereaction was stopped with 2N H₂SO₄, and absorbance was read at 450 nm.

For alanine scanning experiments, expression vectors expressing R1-R14of FIG. 28A were produced by cloning alanine substituted AITR constructs(having a series of individual alanine substitution) into pelFexpression vector, a derivative of pCEP4, in frame with the initiationATG of the N-terminal human PAI leader sequence and the C-terminalpoly-histidine tag. All plasmid DNAs were transient transfected intoExpi293 cells using ExpiFectamine293 Transfection kit (Invitrogen,Carlsbad, Calif., USA). After 7 days, cell culture supernatants werecollected and purified by affinity chromatography using Ni-SepharoseHigh Performance (GE healthcare, Uppsala, Sweden). The expression of theHis-tag fusion protein constructs (R1-R14 of FIG. 28A) was confirmed byWestern blot analysis using 12% SDS-polyacrylamide gel electrophoresis(SDS-PAGE) and coomassie blue staining and anti-His (1:2500; R&DSystems, Minneapolis, Minn., USA) immunoblotting.

For assessing binding of the anti-AITR antibodies IRTCA-A (A27), H1F1,H1F1M69 and H1F1M74 to the His-tag fusion protein constructs R1-R14 ofFIG. 28A, containing alanine substitutions, Western blot and ELISAanalyses similar to those described above for GST-fusion proteinconstructs were performed, but anti-Human IgG-HRP (1:5000; JacksonLaboratories, Bar Harbor, Me.) or anti-His (1:5000; R&D Systems,Minneapolis, Minn., USA) was used as the secondary antibody for ELISAanalysis.

As shown in FIG. 27B, Western blot and ELISA analysis showed binding ofthe anti-AITR antibodies IRTCA-A, H1F1, M69 and M74 to the GST-fusionproteins R1 and R2. Molecular sizes are indicated on the left and eachmutant is indicated by the numbers on the top. Data are representativeof three independent experiments, *p<0.05.

Based on the Western blot and ELISA analyses above, the binding epitopefor IRTCA-A (A27), H1F1, H1F1M69, and H1F1M74 is within the amino acidsequences of AA56 and AA65 of the extracellular domain of AITR, as shownin FIG. 27C.

Alanine scanning of the AITR epitopes was performed by first generatingfourteen His-fusion proteins of alanine substituted AITR proteins,R1-R14, shown in FIG. 28A. Each of the residues constituting the epitoperegion determined by the epitope mapping experiment described in FIGS.27A-C was individually mutated to alanine. This alanine scanning wascarried out with entire ECD of AITR. One to two amino acids were addedat both sides of epitope in the scanning. As shown in FIG. 28B, showedbinding of IRTCA-A (A27), H1F1, H1F1M69, and H1F1M74 to alaninesubstituted AITR fusion proteins R1-R7 and R13-R14, as determined byWestern blot (FIG. 28B, four lower left panels) and ELISA (FIG. 28B,four lower right panels) analysis. Molecular sizes are indicated on theleft and each mutant is indicated by the numbers on the top. Data arerepresentative of three independent experiments, *p<0.05.

Based on the western blot and ELISA analysis of FIG. 28B, the coresequences of epitope for IRTCA-A (A27), H1F1, H1F1M69, and H1F1M74 werefurther narrowed to the 5 amino acid sequences CCTTC, as shown in FIG.28C.

The amino acid sequences of the epitope and the core amino acid residuesfor IRTCA-A, H1F1, M69 or M74 are shown in Table 14.

TABLE 14 10 amino acid binding epitope of Core amino acid mAb AITRresidues IRTCA-A HCGDPCCTTC CCTTC (SEQ ID NO: 19) (SEQ ID NO: 27) H1F1HCGDPCCTTC CCTTC (SEQ ID NO: 19) (SEQ ID NO: 27) H1F1.M69 HCGDPCCTTCCCTTC (H1F1.H2.J1/ (SEQ ID NO: 19) (SEQ ID NO: 27) H1F1.L3.J1) H1F1.M74HCGDPCCTTC CCTTC (H1F1.H8/ (SEQ ID NO: 19) (SEQ ID NO: 27) H1F1.L3.J2)

Example 17: Determination of Epitopes for AITR mAbs A35 and A41

In this example, anti-AITR antibodies A35 and A41 (along with IRTCA-A,already shown above in Example 16) were tested for their ability to bindto GST fusion protein constructs containing overlapping portions of theextracellular domain of AITR (R1-R12 of FIG. 29A).

Twelve GST fusion protein constructs containing overlapping portions ofthe extracellular domain of AITR (R1-R12, shown in FIG. 29A) wereproduced and Coomassie blue staining of recombinant R1 to R12 proteinswas performed to confirm the presence of the GST-fusion proteins (FIG.29B, upper left panel). Molecular size markers are shown on the left.Western blot analysis showed that each of the three mAbs binds to uniquesets of deletion mutants (A35 to R1-4 and R8-11; A41 to R1-6 and R8-9).Based on the Western blot analysis shown in FIG. 29B, the bindingepitope for IRTCA-A (A27), A35, and A41 were identified as shown in FIG.29C.

Alanine scanning of the AITR epitopes (identified by the epitope mappingexperiment of FIGS. 29A-C) was performed by first generating His-fusionproteins of alanine substituted AITR proteins based on the identifiedthree epitope regions shown in FIG. 30A. Each of the residuesconstituting the epitope region determined by the epitope mappingexperiment described in FIGS. 29A-C was individually mutated to alanine.This alanine scanning was carried out with entire ECD of AITR. One totwo amino acids were added to both sides of epitope in the scanning. Asshown in FIG. 30B, Western blot analysis of the alanine-mutated AITR-Hisfusion proteins was performed using one of the three anti-AITRantibodies and a secondary anti-human IgG antibody. Alanine-substitutedAITR protein was detected by anti-His antibody (upper rows). Reactivityof A41, A35 and IRTCA-A to the alanine-substituted AITR was determined(lower rows). As shown in FIG. 30C, ELISA analysis confirmed thefindings of the Western blot analysis of FIG. 30B.

Based on the Western blot analysis above, the core amino acid residuesof epitopes for A41 and A35 were further narrowed to the amino acidsequences CC SEW and CCRVH, respectively. The amino acid sequences ofthe 10-11 amino acid residue epitope and the core amino acid residuesfor A41, A35 or IRTCA-A are shown in Table 15.

TABLE 15 10 amino acid binding Core amino acid mAb epitope of AITRresidues A35 ECCSEWDCMC CCSEW (SEQ ID NO: 28) (SEQ ID NO: 29) A41GTDARCCRVHT CCRVH (SEQ ID NO: 30) (SEQ ID NO: 31)

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the claims.

1.-57. (canceled)
 58. A nucleic acid molecule encoding anIFN-gamma-Inducible Regulatory T Cell Convertible Anti-Cancer (IRTCA)antibody or antigen-binding fragment, comprising: a) a heavy chain CDR1comprising SEQ ID NO: 8, a heavy chain CDR2 comprising SEQ ID NO: 25,and a heavy chain CDR3 comprising SEQ ID NO: 16; and b) a light chainCDR1 comprising SEQ ID NO: 11, a light chain CDR2 comprising SEQ ID NO:12, and a light chain CDR3 comprising SEQ ID NO:
 18. 59. The nucleicacid molecule of claim 58, wherein the nucleic acid further encodes forheavy chain variable region comprising SEQ ID NO: 20 and light chainvariable region comprising SEQ ID NO:
 23. 60. A recombinant vectorcomprising the nucleic acid molecule of claim
 58. 61. A recombinantvector comprising the nucleic acid molecule of claim
 59. 62. A host cellcomprising the recombinant vector of claim
 60. 63. A host cellcomprising the recombinant vector of claim
 61. 64. The host cell ofclaim 62, wherein the host cell is selected from a bacterial, yeast,insect, or mammalian cell.
 65. The host cell of claim 64, wherein thehost cell is selected from the group consisting of E. coli, P. pastoris,Sf9, COS, HEK293, Expi293, CHO-S, CHO-DG44, CHO-K1, and a mammalianlymphocyte.
 66. A nucleic acid molecule encoding an IFN-gamma-InducibleRegulatory T Cell Convertible Anti-Cancer (IRTCA) antibody orantigen-binding fragment, comprising: a) a heavy chain CDR1 comprisingSEQ ID NO: 24, a heavy chain CDR2 comprising SEQ ID NO: 25, and a heavychain CDR3 comprising SEQ ID NO: 16; and b) a light chain CDR1comprising SEQ ID NO: 11, a light chain CDR2 comprising SEQ ID NO: 12,and a light chain CDR3 comprising SEQ ID NO:
 13. 67. The nucleic acidmolecule of claim 66, wherein the nucleic acid further encodes for heavychain variable region comprising SEQ ID NO: 21, and light chain variableregion SEQ ID NO:
 22. 68. A recombinant vector comprising the nucleicacid molecule of claim
 66. 69. A recombinant vector comprising thenucleic acid molecule of claim
 67. 70. A host cell comprising therecombinant vector of claim
 68. 71. A host cell comprising therecombinant vector of claim
 69. 72. The host cell of claim 70, whereinthe host cell is selected from a bacterial, yeast, insect, or mammaliancell.
 73. The host cell of claim 72, wherein the host cell is selectedfrom the group consisting of E. coli, P. pastoris, Sf9, COS, HEK293,Expi293, CHO-S, CHO-DG44, CHO-K1, and a mammalian lymphocyte.
 74. Amethod of inducing an immune response in a subject in need thereof,comprising: administering to the subject a composition that comprises ordelivers the IRTCA nucleic acid of claim
 58. 75. A method of enhancingan immune response or increasing the activity of an immune cell in asubject in need thereof, comprising: administering to the subject acomposition that comprises or delivers the IRTCA nucleic acid of claim58.
 76. A method of inducing an immune response in a subject in needthereof, comprising: administering to the subject a composition thatcomprises or delivers the IRTCA nucleic acid of claim
 66. 77. A methodof enhancing an immune response or increasing the activity of an immunecell in a subject in need thereof, comprising: administering to thesubject a composition that comprises or delivers the IRTCA nucleic acidof claim 66.